Modulators of body weight, corresponding nucleic acids and proteins

ABSTRACT

Modulators of weight, including, for example, two isoforms of murine and human ob polypeptides, are provided, as well as diagnostic and therapeutic uses and methods comprising such. Also provided are nucleotide sequences, degenerate variations thereof, and proteins expressed by such.

This application is a divisional of U.S. application Ser. No.10/780,295, filed Feb. 17, 2004, now U.S. Pat. No. 7,521,258, which is adivisional of U.S. application Ser. No. 09/316,393, filed May 21, 1999,now U.S. Pat. No. 6,734,160, which is a divisional of U.S. applicationSer. No. 08/292,345, filed Aug. 17, 1994, now U.S. Pat. No. 6,001,968,each of which is incorporated by reference in its entirety.

The research leading to the present inventions was funded in part byGrant No. DK 41096 from the National Institutes of Health. Thegovernment may have certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to the control of body weight ofmammals including animals and humans, and more particularly to materialsidentified herein as modulators of weight, and to the diagnostic andtherapeutic uses to which such modulators may be put.

BACKGROUND OF THE INVENTION

Obesity, defined as an excess of body fat relative to lean body mass, isassociated with important psychological and medical morbidities, thelatter including hypertension, elevated blood lipids, and Type II ornon-insulin-dependent diabetes melitis (NIDDM). There are 6-10 millionindividuals with NIDDM in the U.S., including 18% of the population of65 years of age (Harris et al., 1987). Approximately 45% of males and70% of females with NIDDM are obese, and their diabetes is substantiallyimproved or eliminated by weight reduction (Harris, 1991). As describedbelow, both obesity and NIDDM are strongly heritable, though thepredisposing genes have not been identified. The molecular genetic basisof these metabolically related disorders is an important, poorlyunderstood problem.

The assimilation, storage, and utilization of nutrient energy constitutea complex homeostatic system central to survival of metazoa. Amongland-dwelling mammals, storage in adipose tissue of large quantities ofmetabolic fuel as triglycerides is crucial for surviving periods of fooddeprivation. The need to maintain a fixed level of energy stores withoutcontinual alterations in the size and shape of the organism requires theachievement of a balance between energy intake and expenditure. However,the molecular mechanisms that regulate energy balance remain to beelucidated. The isolation of molecules that transduce nutritionalinformation and control energy balance will be critical to anunderstanding of the regulation of body weight in health and disease.

An individual's level of adiposity is, to a large extent, geneticallydetermined. Examination of the concordance rates of body weight andadiposity amongst mono- and dizygous twins or adoptees and theirbiological parents have suggested that the heritability of obesity(0.4-0.8) exceeds that of many other traits commonly thought to have asubstantial genetic component, such as schizophrenia, alcoholism, andatherosclerosis (Stunkard et al., 1990). Familial similarities in ratesof energy expenditure have also been reported (Bogardus et al., 1986).Genetic analysis in geographically delimited populations has suggestedthat a relatively small number of genes may account for the 30%-50% ofvariance in body composition (Moll et al., 1991). However, none of thegenes responsible for obesity in the general population have beengenetically mapped to a definite chromosomal location.

Rodent models of obesity include seven apparently single-gene mutations.The most intensively studied mousse obesity mutations are the ob (obese)and db (diabetes) genes. When present on the same genetic strainbackground, ob and db result in indistinguishable metabolic andbehavioral phenotypes, suggesting that these genes may function in thesame physiologic pathway (Coleman, 1978). Mice homozygous for eithermutation are hyperphagic and hypometabolic, leading to an obesephenotype that is notable at one month of age. The weight of theseanimals tends to stabilize at 60-70 g (compared with 30-35 g in controlmice). ob and db animals manifest a myriad of other hormonal andmetabolic changes that have made it difficult to identify the primarydefect attributable to the mutation (Bray et al., 1989).

Each of the rodent obesity models is accompanied by alterations incarbohydrate metabolism resembling those in Type II diabetes in man. Insome cases, the severity of the diabetes depends in part on thebackground mouse strain (Leiter, 1989). For both ob and db, congenicC57BL/Ks mice develop a severe diabetes with ultimate β cell necrosisand islet atrophy, resulting in a relative insulinopenia. Conversely,congenic C57BL/6J ob and db mice develop a transient insulin-resistantdiabetes that is eventually compensated by β cell hypertrophy resemblinghuman Type II diabetes.

The phenotype of ob and db mice resembles human obesity in ways otherthan the development of diabetes—the mutant mice eat more and expendless energy than do lean controls (as do obese humans). This phenotypeis also quite similar to that seen in animals with lesions of theventromedial hypothalamus, which suggests that both mutations mayinterfere with the ability to properly integrate or respond tonutritional information within the central nervous system. Support forthis hypothesis comes from the results of parabiosis experiments(Coleman, 1973) that suggest ob mice are deficient in a circulatingsatiety factor and that db mice are resistant to the effects of the obfactor (possibly due to an ob receptor defect). These experiments haveled to the conclusion that obesity in these mutant mice may result fromdifferent defects in an afferent loop and/or integrative center of thepostulated feedback mechanism that controls body composition.

Using molecular and classical genetic markers, the ob and db genes havebeen mapped to proximal chromosome 6 and midchromosome 4, respectively(Bahary et al., 1990; Friedman et al., 1991b). In both cases, themutations map to regions of the mouse genome that are syntonic withhuman, suggesting that, if there are human homologs of ob and db, theyare likely to map, respectively, to human chromosomes 7q and 1p. Defectsin the db gene may result in obesity in other mammalian species: ingenetic crosses between Zucker falfa rats and Brown Norway+/+ rats, thefa mutation (rat chromosome 5) is flanked by the same loci that flank dbin mouse (Truett et al., 1991).

Because of the myriad factors that seem to impact body weight, it isdifficult to speculate as to which of these factors, and moreparticularly, which homeostatic mechanism is actually primarilydeterminative. Nonetheless, the apparent connection between the ob geneand the extent and characteristics of obesity have prompted the furtherinvestigation and elucidation that is reflected by the presentapplication. It is the identification of the sequence of the gene andcorresponding peptide materials, to which the present inventionfollowing below directs itself.

The citation of any reference herein should not be construed as anadmission that such reference is prior art to the instant invention.Full citations of references cited by author and year are found at theend of the specification.

SUMMARY OF THE INVENTION

In its broadest aspect, the present invention relates to the elucidationand discovery of nucleotide sequences, and proteins putatively expressedby such nucleic acids or degenerate variations thereof, that demonstratethe ability to participate in the control of mammalian body weight. Thenucleotide sequences in object are believed to represent the genescorresponding to the murine and human ob gene, that is postulated toplay a critical role in the regulation of body weight and adiposity.Preliminary data, presented herein, suggests that the polypeptideproduct of the gene in question functions as a hormone.

In a first instance, the modulators of the present invention comprisenucleic acid molecules, including recombinant DNA molecules or clonedgenes, or degenerate variants thereof, which encode polypeptidesthemselves serving as modulators of weight control as hereinafterdefined, or conserved variants or fragments thereof, which polypeptidespossess amino acid sequences such as set forth in FIG. 3 (SEQ ID NO:2),FIG. 4 (SEQ ID NO:4), FIG. 5 (SEQ ID NO:5) and FIG. 6 (SEQ ID NO:6). Inspecific embodiments, amino acid sequences for two variants of murineand human ob polypeptides are provided. Both polypeptides are found in aform with glutamine 49 deleted, which may result from mRNA splicing.

The nucleic acid molecules, recombinant DNA molecules, or cloned genes,may have the nucleotide sequences or may be complementary to DNAsequences shown in FIG. 1 (SEQ ID NO:1) and FIG. 2 (SEQ ID NO:3).Accordingly, the present invention also relates to the identification ofa gene having a nucleotide sequence selected from the sequences of FIG.1 (SEQ ID NO:1) and FIG. 2 (SEQ ID NO:3) herein, and degeneratevariants, allelic variations, and like cognate molecules.

A nucleic acid molecule of the invention can be DNA or RNA, includingsynthetic variants thereof having phosphate or phosphate analog, e.g.,thiophosphate, bonds. Both single stranded and double stranded sequencesare contemplated herein.

The present invention further provides nucleic acid molecules for use asmolecular probes, or as primers for polymerase chain reaction (PCR)amplification, i.e., synthetic or natural oligonucleotides having asequence corresponding to a portion of the sequences shown in FIG. 1(SEQ ID NO:1) and FIG. 2 (SEQ ID NO:3). In particular, the inventioncontemplates a nucleic acid molecule having at least about 10nucleotides, wherein a sequence of the nucleic acid molecule correspondsto a nucleotide sequence of the same number of nucleotides in thenucleotide sequences of FIG. 1 (SEQ ID NO:1) or FIG. 2 (SEQ ID NO:3), ora sequence complementary thereto. More preferably, the nucleic acidsequence of the molecule has at least 15 nucleotides. Most preferably,the nucleic acid sequence has at least 20 nucleotides. In an embodimentof the invention in which the oligonucleotide is a probe, theoligonucleotide is detectably labeled, e.g., with a radionuclide (suchas ³²P), or an enzyme.

In further aspects, the present invention provides a cloning vector,which comprises the nucleic acids of the invention; and a bacterial,insect, or a mammalian expression vector, which comprises the nucleicacid molecules of the invention, operatively associated with anexpression control sequence. Accordingly, the invention further relatesto a bacterial cell or a mammalian transfected or transformed with anappropriate expression vector, and correspondingly, to the use of theabove mentioned constructs in the preparation of the modulators of theinvention.

All of the foregoing materials are to be considered herein as modulatorsof body weight and fat composition, and as such, may be used in avariety of contexts. Specifically, the invention contemplates bothdiagnostic and therapeutic applications, as well as certain agriculturalapplications, all contingent upon the use of the modulators definedherein, including both nucleic acid molecules and peptides. Moreover,the modulation of body weight carries specific therapeutic implicationsand benefits, in that conditions where either obesity or, conversely,cachexia represent undesired bodily conditions, can be remedied by theadministration of one or more of the modulators of the presentinvention.

Thus, a method for modulating body weight of a mammal is proposed thatcomprises controlling the expression of the protein encoded by a nucleicacid having nucleotide sequence selected from the sequence of FIG. 1(SEQ ID NO:1), the sequence of FIG. 2 (SEQ ID NO:3) and degenerate andallelic variants thereof. Such control may be effected by theintroduction of the nucleotides in question by gene therapy into fatcells of the patient or host to control or reduce obesity. Conversely,the preparation and administration of antagonists to the nucleotides,such as anti-sense molecules, would be indicated and pursued in theinstance where conditions involving excessive weight loss, such asanorexia nervosa, cancer, or AIDS are present and under treatment. Suchconstructs would be introduced in similar fashion to the nucleotides,directly into fat cells to effect such changes.

Correspondingly, the proteins defined by FIGS. 3, 4, 5, and 6 (SEQ IDNO:1, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6), conserved variants,active fragments thereof, and cognate small molecules could beformulated for direct administration for therapeutic purposes, to effectreduction or control of excessive body fat or weight gain.Correspondingly, antibodies and other antagonists to the stated proteinmaterials could be prepared and similarly administered to achieve theconverse effect. Accordingly, the invention is advantageously directedto a pharmaceutical composition comprising an ob polypeptide of theinvention, or alternatively an antagonist thereof, in an admixture witha pharmaceutically acceptable carrier or excipient.

The diagnostic uses of the present nucleotides and correspondingpeptides extend to the use of the nucleotides to identify furthermutations of allelic variations thereof, so as to develop a repertoireof active nucleotide materials useful in both diagnostic and therapeuticapplications. In particular, both homozygous and heterozygous mutationsof the nucleotides in question could be prepared that would bepostulated to more precisely quantitate the condition of patients, todetermine the at-risk potential of individuals with regard to obesity.Specifically, heterozygous mutations are presently viewed as associatedwith mild to moderate obesity, while homozygous mutations would beassociated with a more pronounced and severe obese condition.Corresponding DNA testing could then be conducted utilizing theaforementioned ascertained materials as benchmarks, to facilitate anaccurate long term prognosis for particular tendencies, so as to be ableto prescribe changes in either dietary or other personal habits, ordirect therapeutic intervention, to avert such conditions.

The diagnostic utility of the present invention extends to methods formeasuring the presence and extent of the modulators of the invention incellular samples or extracts taken from test subjects, so that both theencoded nucleotide (RNA) and or the levels of protein in such testsamples could be ascertained. Given that the increased activity of thenucleotide and presence of the resulting protein reflect the capabilityof the subject to inhibit obesity, the physician reviewing such resultsin an obese subject would determine that a factor other than dysfunctionwith respect to the presence and activity of the nucleotides of thepresent invention is a cause of the obese condition. Conversely,depressed levels of the nucleotide and or the expressed protein wouldsuggest that such levels must be increased to treat such obesecondition, and an appropriate therapeutic regimen could then beimplemented.

Further, the nucleotides discovered and presented in FIGS. 1 and 2represent cDNA in which, as stated briefly above, is useful in themeasurement of corresponding RNA. Likewise, recombinant protein materialcorresponding to the polypeptides of FIGS. 3 and 4 may be prepared andappropriately labeled, for use, for example, in radioimmunoassays, forexample, for the purpose of measuring fat and or plasma levels of the obprotein.

The invention further directs itself recombinant DNA moleculescomprising the DNA sequences of FIGS. 1 and 2, which molecules are in afurther embodiment operatively linked to an expression control sequence.Suitable expression control sequences may be selected from among thosepresently and generally available and in use. The invention furtherextends to probes prepared from the sequences of FIG. 1 or 2 and tohosts transformed with recombinant DNA molecules prepared in accordancewith the present invention.

Yet further, the present invention contemplates not only theidentification of the nucleotides and corresponding proteins presentedherein, but the elucidation of the receptor to such materials. In suchcontext, the polypeptides of FIGS. 3, 4, 5, and/or 6 could be preparedand utilized to screen an appropriate expression library to isolateactive receptors. The receptor could thereafter be cloned, and thereceptor alone or in conjunction with the ligand could thereafter beutilized to screen for small molecules that may possess like activity tothe modulators herein.

Yet further, the present invention relates to pharmaceuticalcompositions that include certain of the modulators hereof, preferablythe polypeptides whose sequences are presented in SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:5 and SEQ ID NO:6, their antibodies, corresponding smallmolecules exhibiting either antagonism or mimicry, or active fragmentsprepared in formulations for a variety of modes of administration, wheresuch therapy is appropriate. Such formulations would includepharmaceutically acceptable carriers, or other adjuvants as needed, andwould be prepared in effective dosage ranges to be determined by theclinician or the physician in each instance.

Accordingly, it is a principal object of the present invention toprovide modulators of body weight as defined herein in purified form,that exhibit certain characteristics and activities associated withcontrol and variation of adiposity and fat content of mammals.

It is a further object of the present invention to provide methods forthe detection and measurement of the modulators of weight control as setforth herein, as a means of the effective diagnosis and monitoring ofpathological conditions wherein the variation in level of suchmodulators is or may be a characterizing feature.

It is a still further object of the present invention to provide amethod and associated assay system for the screening of substances, suchas drugs, agents and the like, that are potentially effective to eithermimic or inhibit the activity of the modulators of the invention inmammals.

It is a still further object of the present invention to provide amethod for the treatment of mammals to control body weight and fatcontent in mammals, and or to treat certain of the pathologicalconditions of which abnormal depression or elevation of body weight is acharacterizing feature.

It is a still further object of the present invention to prepare geneticconstructs for use in genetic therapeutic protocols and orpharmaceutical compositions for comparable therapeutic methods, whichcomprise or are based upon one or more of the modulators, bindingpartners, or agents that may control their production, or that may mimicor antagonize their activities.

Other objects and advantages will become apparent to those skilled inthe art from a review of the ensuing description which proceeds withreference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleic acid sequence derived for the murine ob gene.The nucleotides are numbered from 1 to 701 with a start site atnucleotide 46 and a termination at nucleotide 550.

FIG. 2 depicts the nucleic acid sequence derived for the human ob gene.The nucleotides are numbered from 1 to 701 with a start site atnucleotide 46 and a termination at nucleotide 550.

FIG. 3 depicts the full deduced amino acid sequence derived for themurine ob gene corresponding to the nucleic acid sequence of FIG. 1. Thenucleotides are numbered from 1 to 167. A signal sequence cleavage siteis located after amino acid 21 (Ala) so that the mature protein extendsfrom amino acid 22 (Val) to amino acid 167 (Cys).

FIG. 4 depicts the full deduced amino acid sequence derived for thehuman ob gene corresponding to the nucleic acid sequence of FIG. 2. Theamino acids are numbered from 1 to 167. A signal sequence cleavage siteis located after amino acid 21 (Ala) so that the mature protein extendsfrom amino acid 22 (Val) to amino acid 167 (Cys).

FIG. 5 depicts the full length amino acid sequence (SEQ ID NO:5) derivedfor the murine ob gene as shown in FIG. 3, but lacking glutamine atposition 49. The nucleotides are numbered from 1 to 166. A signalsequence cleavage site is located after amino acid 21 (Ala) (and thus,before the glutamine 49 deletion) so that the mature protein extendsfrom amino acid 22 (Val) to amino acid 166 (Cys).

FIG. 6 depicts the full deduced amino acid sequence (SEQ ID NO:6)derived for the human ob gene as shown in FIG. 4, but lacking glutamineat position 49. The nucleotides are numbered from 1 to 166. A signalsequence cleavage site is located after amino acid 21 (Ala) (and thus,before the glutamine 49 deletion) so that the mature protein extendsfrom amino acid 22 (Val) to amino acid 166 (Cys).

FIG. 7 presents a physical map of the location of ob in the murinechromosome, and the YAC and P1 cloning maps. “M and N” corresponds toMull and NotI restriction sites. The numbers that are followed byparentheses correspond to individual animals that were recombinant inthe region of ob. Ignore the numbers in parentheses. 39gt, Pax-4, D6Drck13 cp2, and met, refer to locations in the region of ob that bind tothe DNA probes. (A) The top series of lines is a schematic mapcorresponding to a region of the chromosome. (B) The next series oflines corresponds to YACs (yeast artificial chromosomes) from theregion. (C) The bottom lines correspond to P1 clones from the region.

FIG. 8 present a photograph of an ethidium bromide stain of 192independent isolates of the exon trapping experiment that werecharacterized.

FIG. 9 is a photograph of an ethidium bromide stain of PCR-amplifiedclones suspected of carrying ob. Each of the 7 clones that did not carrythe artifact was reamplified using PCR and electrophoresed on a 1%agarose gel in TBE and stained with ethidium bromide. The size markers(far left unnumbered lane) are the commercially available “1 kB ladder”.Lane 1—clone 1D12, containing an “HIV sequence.” Lane 2—clone 1F1, anovel clone outside of the ob region. Lane 3—clone 1H3. Lane 4—clone2B2, which is the identical to 1F1. Lane 5—clone 2G7, which contains anob exon. Lane 6—clone 2G11, which is identical to 1F1. Lane 7—clone 2H1,which does not contain an insert.

FIG. 10 presents the sequence of the 2G7 clone, which includes an exoncoding for a part of the ob gene. The primer sequences used to amplifythis exon are boxed in the figure.

FIG. 11 is a Northern blot of mRNA from different organs of the mouseusing PCR labeled 2G7 as a probe. Ten μg of total RNA from each of thetissues was electrophoresed on an agarose gel with formaldehyde. Theprobe was hybridized at 65° C. in Rapid Hybe (Amersham).

FIG. 12 is an ethidium bromide stain from an RT PCR reaction on fat cellRNA from each of the mouse strains listed. Total RNA for each sample wasreverse transcribed using oligo dT and reverse transcriptase, and theresulting single stranded cDNA was PCR amplified with the 2G7 primers(lower bands) or actin primers (upper bands). The products were run on a1% agarose TBE gel.

FIG. 13 is a Northern analysis corresponding to the data in FIG. 12. Tenμg of fat cell RNA were run out and probed with the PCR labeled 2G7probe as in FIG. 11, above.

FIG. 14 is a Northern analysis of 2J animals and control animals thatconfirms the absence of the ob mRNA from 2J animals. The Northernanalysis was performed as in FIGS. 11 and 13. In this case, the controlRNA was ap2, a fat specific transcript. There is no significance to thevarying density of the ap2 bands.

FIG. 15 compares the DNA sequence of the C57BL/6J and the ob 1J mice.The chromatogram shown is the output of a DNA sequencing reaction usingan Applied Biosystem 373A automated DNA sequencer.

FIG. 16 is a genomic southern blot of genomic DNA from each of the mousestrains listed. Approximately 10 μg of DNA (derived from genomic DNAprepared from liver, kidney or spleen) was restriction digested with therestriction enzyme indicated. The DNA was then electrophoresed in a 1%agarose TBE gel and probed with PCR labeled 2G7.

FIG. 17 is a Southern blot of BglII digests of genomic DNA from theprogeny of an ob^(2J)/+ ob^(2J)/+ cross.

FIG. 18 is a Southern blot of EcoRI digested DNA from the specieslisted, using 2G7 as a probe. The restricted DNA was run on a 1% agaroseTBE gel, and transferred to an imobilon membrane for probing. The filterwas hybridized at 65° C. in Rapid Hype buffer, and washed with 2×SSC, 2%SDS at 65° C. twice for 30 minutes each.

FIG. 19 presents the expression cloning region of vector pET-15b(Novagen).

DETAILED DESCRIPTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

The term “body weight modulator”, “modulator”, “modulators”, and anyvariants not specifically listed, may be used herein interchangeably,and as used throughout the present application and claims refers in oneinstance to both nucleotides and to proteinaceous material, the latterincluding both single or multiple proteins. More specifically, theaforementioned terms extend to the nucleotides and to the DNA having thesequences described herein and presented in FIG. 1 (SEQ ID NO:1), andFIG. 2 (SEQ ID NO:3). Likewise, the proteins having the amino acidsequence data described herein and presented in FIG. 3 (SEQ ID NO:2),and FIG. 4 (SEQ ID NO:4) are likewise contemplated, as are the profileof activities set forth with respect to all materials both herein and inthe claims. Accordingly, nucleotides displaying substantially equivalentor altered activity are likewise contemplated, including substantiallyhomologous analogs and allelic variations. Likewise, proteins displayingsubstantially equivalent or altered activity, including proteinsmodified deliberately, as for example, by site-directed mutagenesis, oraccidentally through mutations in hosts that produce the modulators arelikewise contemplated.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo, i.e.,capable of replication under its own control.

A “vector” is a replicon, such as a plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “cassette” refers to a segment of DNA that can be inserted into avector at specific restriction sites. The segment of DNA encodes apolypeptide of interest, and the cassette and restriction sites aredesigned to ensure insertion of the cassette in the proper reading framefor transcription and translation.

“Heterologous” DNA refers to DNA not naturally located in the cell, orin a chromosomal site of the cell. Preferably, the heterologous DNAincludes a gene foreign to the cell.

A cell has been “transfected” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. A cell has been “transformed”by exogenous or heterologous DNA when the transfected DNA effects aphenotypic change. Preferably, the transforming DNA should be integrated(covalently linked) into chromosomal DNA making up the genome of thecell.

A “clone” is a population of cells derived from a single cell or commonancestor by mitosis.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”) in either singlestranded form, or a double-stranded helix. Double stranded DNA-DNA,DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary or quaternary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acids,low stringency hybridization conditions, corresponding to a T_(m) of55°, can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide;or 30% formamide, 5×SSC, 0.5% SDS). Moderate stringency hybridizationconditions correspond to a higher T_(m), e.g., 40% formamide, with 5× or6×SCC. High stringency hybridization conditions correspond to thehighest T_(m), e.g., 50% formamide, 5× or 6×SCC. Hybridization requiresthat the two nucleic acids contain complementary sequences, althoughdepending on the stringency of the hybridization, mismatches betweenbases are possible. The appropriate stringency for hybridizing nucleicacids depends on the length of the nucleic acids and the degree ofcomplementation, variables well known in the art. The greater the degreeof similarity or homology between two nucleotide sequences, the greaterthe value of T_(m) for hybrids of nucleic acids having those sequences.The relative stability (corresponding to higher T_(m)) of nucleic acidhybridizations decreases in the following order: RNA:RNA, DNA:RNA,DNA:DNA. For hybrids of greater than 100 nucleotides in length,equations for calculating T_(m) have been derived (see Sambrook et al.,supra, 9.50-0.51). For hybridization with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (seeSambrook et al., supra, 11.7-11.8). Preferably a minimum length for ahybridizable nucleic acid is at least about 10 nucleotides; morepreferably at least about 15 nucleotides; most preferably the length isat least about 20 nucleotides.

“Homologous recombination” refers to the insertion of a foreign DNAsequence of a vector in a chromosome. Preferably, the vector targets aspecific chromosomal site for homologous recombination. For specifichomologous recombination, the vector will contain sufficiently longregions of homology to sequences of the chromosome to allowcomplementary binding and incorporation of the vector into thechromosome. Longer regions of homology, and greater degrees of sequencesimilarity, may increase the efficiency of homologous recombination.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.The boundaries of the coding sequence are determined by a start codon atthe 5′ (amino) terminus and a translation stop codon at the 3′(carboxyl) terminus. A coding sequence can include, but is not limitedto, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNAsequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNAsequences. If the coding sequence is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell. Ineukaryotic cells, polyadenylation signals are control sequences.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced and translated into the protein encoded by the coding sequence.

A “signal sequence” is included at the beginning of the coding sequenceof a protein to be expressed on the surface of a cell. This sequenceencodes a signal peptide, N-terminal to the mature polypeptide, thatdirects the host cell to translocate the polypeptide. The term“translocation signal sequence” is also used herein to refer to thissort of signal sequence. Translocation signal sequences can be foundassociated with a variety of proteins native to eukaryotes andprokaryotes, and are often functional in both types of organisms.

A DNA sequence is “operatively linked” to an expression control sequencewhen the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the DNA sequence to be expressed and maintaining thecorrect reading frame to permit expression of the DNA sequence under thecontrol of the expression control sequence and production of the desiredproduct encoded by the DNA sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted upstream (5′) of andin reading frame with the gene.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

The term “standard hybridization conditions” refers to salt andtemperature conditions substantially equivalent to 5×SSC and 65° C. forboth hybridization and wash.

A molecule is “antigenic” when it is capable of specifically interactingwith an antigen recognition molecule of the immune system, such as animmunoglobulin (antibody) or T cell antigen receptor. An antigenicpolypeptide contains at least about 5, and preferably at least about 10,amino acids. An antigenic portion of a molecule can be that portion thatis immunodominant for antibody or T cell receptor recognition, or it canbe a portion used to generate an antibody to the molecule by conjugatingthe antigenic portion to a carrier molecule for immunization. A moleculethat is antigenic need not be itself immunogenic, i.e., capable ofeliciting an immune response without a carrier.

An “antibody” is any immunoglobulin, including antibodies and fragmentsthereof, that binds a specific epitope. The term encompasses polyclonal,monoclonal, and chimeric antibodies, the last mentioned described infurther detail in U.S. Pat. Nos. 4,816,397 and 4,816,567, as well asantigen binding portions of antibodies, including Fab, F(ab′)₂ and Fr(including single chain antibodies). Accordingly, the phrase “antibodymolecule” in its various grammatical forms as used herein contemplatesboth an intact immunoglobulin molecule and an immunologically activeportion of an immunoglobulin molecule containing the antibody combiningsite. An “antibody combining site” is that structural portion of anantibody molecule comprised of heavy and light chain variable andhypervariable regions that specifically binds antigen.

Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contains the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′)₂ and F(v), which portionsare preferred for use in the therapeutic methods described herein.

Fab and F(ab′)₂ portions of antibody molecules are prepared by theproteolytic reaction of papain and pepsin, respectively, onsubstantially intact antibody molecules by methods that are well-known.See for example, U.S. Pat. No. 4,342,566 to Theofilopolous et al. Fab′antibody molecule portions are also well-known and are produced fromF(ab′)₂ portions followed by reduction of the disulfide bonds linkingthe two heavy chain portions as with mercaptoethanol, and followed byalkylation of the resulting protein mercaptan with a reagent such asiodoacetamide. An antibody containing intact antibody molecules ispreferred herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

A composition comprising “A” (where “A” is a single protein, DNAmolecule, vector, recombinant host cell, etc.) is substantially free of“B” (where “B” comprises one or more contaminating proteins, DNAmolecules, vectors, etc., but excluding racemic forms of A) when atleast about 75% by weight of the proteins, DNA, vectors (depending onthe category of species to which A and B belong) in the composition is“A”. Preferably, “A” comprises at least about 90% by weight of the A+Bspecies in the composition, most preferably at least about 99% byweight. It is also preferred that a composition, which is substantiallyfree of contamination, contain only a single molecular weight specieshaving the activity or characteristic of the species of interest.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human. Preferably, asused herein, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the compound isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water or aqueous solution saline solutions and aqueousdextrose and glycerol solutions are preferably employed as carriers,particularly for injectable solutions. Suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to reduce by at least about 15 percent, preferably byat least 50 percent, more preferably by at least 90 percent, and mostpreferably prevent, a clinically significant deficit in the activity,function and response of the host. Alternatively, a therapeuticallyeffective amount is sufficient to cause an improvement in a clinicallysignificant condition in the host.

The term “adjuvant” refers to a compound or mixture that enhances theimmune response to an antigen. An adjuvant can serve as a tissue depotthat slowly releases the antigen and also as a lymphoid system activatorthat non-specifically enhances the immune response (Hood et al.,Immunology, Second Ed., 1984, Benjamin/Cummings: Menlo Park, Calif., p.384). Often, a primary challenge with an antigen alone, in the absenceof an adjuvant, will fail to elicit a humoral or cellular immuneresponse. Adjuvants include, but are not limited to, complete Freund'sadjuvant, incomplete Freund's adjuvant, saponin, mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Preferably, the adjuvant is pharmaceutically acceptable.

In its primary aspect, the present invention is directed to theidentification of materials that function as modulators of mammalianbody weight. In particular, the invention concerns the isolation,purification and sequencing of certain nucleic acids that correspond tothe ob gene in both mice and humans, as well as the correspondingpolypeptides expressed by these nucleic acids. The invention thuscomprises the discovery of nucleic acids having the nucleotide sequencesset forth in FIG. 1 (SEQ ID NO:1) and FIG. 2 (SEQ ID NO:3), and todegenerate variants, alleles and fragments thereof, all possessing theactivity of modulating body weight and adiposity. The correspondence ofthe present nucleic acids to the ob gene portends their significantimpact on conditions such as obesity as well as other maladies anddysfunctions where abnormalities in body weight are a contributoryfactor. The invention extends to the proteins expressed by the nucleicacids of the invention, and particularly to those proteins set forth inFIG. 3 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), FIG. 5 (SEQ ID NO:5), andFIG. 6 (SEQ ID NO:6), as well as conserved variants, active fragments,and cognate small molecules.

In particular, the present invention contemplates that naturallyoccurring fragments of the ob polypeptide may be important. The peptidesequence includes a number of sites that are frequently the target forproteolytic cleavage, e.g., arginine residues. It is possible that thefull length polypeptide may be cleaved at one or more such sites to formbiologically active fragments. Such biologically active fragments mayeither agonize or antagonize the functional activity of the obpolypeptide to reduce body weight.

As discussed earlier, the weight control modulator peptides or theirbinding partners or other ligands or agents exhibiting either mimicry orantagonism to them or control over their production, may be prepared inpharmaceutical compositions, with a suitable carrier and at a strengtheffective for administration by various means to a patient experiencingabnormal fluctuations in body weight or adiposity, either alone or aspart of an adverse medical condition such as cancer or AIDS, for thetreatment thereof. A variety of administrative techniques may beutilized, among them parenteral techniques such as subcutaneous,intravenous and intraperitoneal injections, catheterizations and thelike. Average quantities of the recognition factors or their subunitsmay vary and in particular should be based upon the recommendations andprescription of a qualified physician or veterinarian.

Also, antibodies including both polyclonal and monoclonal antibodies,and drugs that modulate the production or activity of the weight controlmodulators recognition factors and/or their subunits may possess certaindiagnostic applications and may for example, be utilized for the purposeof detecting and/or measuring conditions where abnormalities in bodyweight are or may be likely to develop. For example, the modulatorpeptides or their active fragments may be used to produce bothpolyclonal and monoclonal antibodies to themselves in a variety ofcellular media, by known techniques such as the hybridoma techniqueutilizing, for example, fused mouse spleen lymphocytes and myelomacells. These techniques are described in detail below. Likewise, smallmolecules that mimic or antagonize the activity(ies) of the receptorrecognition factors of the invention may be discovered or synthesized,and may be used in diagnostic and/or therapeutic protocols.

Panels of monoclonal antibodies produced against modulator peptides canbe screened for various properties; i.e., isotype, epitope, affinity,etc. Of particular interest are monoclonal antibodies that neutralizethe activity of the modulator peptides. Such monoclonals can be readilyidentified in activity assays for the weight modulators. High affinityantibodies are also useful when immunoaffinity purification of native orrecombinant modulator is possible.

Preferably, the anti-modulator antibody used in the diagnostic andtherapeutic methods of this invention is an affinity purified polyclonalantibody. More preferably, the antibody is a monoclonal antibody (mAb).In addition, it is preferable for the anti-modulator antibody moleculesused herein be in the form of Fab, Fab′, F(ab′)₂ or F(v) portions ofwhole antibody molecules.

As suggested earlier, a diagnostic method useful in the presentinvention comprises examining a cellular sample or medium by means of anassay including an effective amount of an antagonist to a modulatorprotein, such as an anti-modulator antibody, preferably anaffinity-purified polyclonal antibody, and more preferably a mAb. Inaddition, it is preferable for the anti-modulator antibody moleculesused herein be in the form of Fab, Fab′, F(ab′)₂ or F(v) portions orwhole antibody molecules. As previously discussed, patients capable ofbenefiting from this method include those suffering from cancer, AIDS,obesity or other condition where abnormal body weight is acharacteristic or factor. Methods for isolating the modulator andinducing anti-modulator antibodies and for determining and optimizingthe ability of anti-modulator antibodies to assist in the examination ofthe target cells are all well-known in the art.

The nucleic acids contemplated by the present invention extend asindicated, to other nucleic acids that code on expression for peptidessuch as those set forth in FIG. 3 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4),FIG. 5 (SEQ ID NO:5), and FIG. 6 (SEQ ID NO:6) herein. Accordingly,while specific DNA has been isolated and sequenced in relation to the obgene, any animal cell potentially can serve as the nucleic acid sourcefor the molecular cloning of a gene encoding the peptides of theinvention. The DNA may be obtained by standard procedures known in theart from cloned DNA (e.g., a DNA “library”), by chemical synthesis, bycDNA cloning, or by the cloning of genomic DNA, or fragments thereof,purified from the desired cell (See, for example, Sambrook et al., 1989,supra; Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRLPress, Ltd., Oxford, U.K. Vol. I, II). Clones derived from genomic DNAmay contain regulatory and intron DNA regions in addition to codingregions; clones derived from cDNA will not contain intron sequences.Whatever the source, the gene should be molecularly cloned into asuitable vector for propagation of the gene.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired gene. The DNA may becleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired ob or ob-like gene may be accomplishedin a number of ways. For example, if an amount of a portion of a ob orob-like gene or its specific RNA, or a fragment thereof, is availableand can be purified and labeled, the generated DNA fragments may bescreened by nucleic acid hybridization to the labeled probe (Benton andDavis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc. Natl.Acad. Sci. U.S.A. 72:3961). The present invention provides such nucleicacid probes, which can be conveniently prepared from the specificsequences disclosed herein, e.g., a hybridizable probe having anucleotide sequence corresponding to at least a 10, and preferably a 15,nucleotide fragment of the sequences depicted in FIG. 1 (SEQ ID NO:1) orFIG. 2 (SEQ ID NO:3). Preferably, a fragment is selected that is highlyunique to the modulator peptides of the invention. Those DNA fragmentswith substantial homology to the probe will hybridize. As noted above,the greater the degree of homology, the more stringent hybridizationconditions can be used. In a specific embodiment, low stringencyhybridization conditions are used to identify a homologous modulatorpeptide. However, in a preferred aspect, a nucleic acid encoding amodulator peptide of the invention will hybridize to a nucleic acidhaving a nucleotide sequence such as depicted in FIG. 1 (SEQ ID NO: 1)or FIG. 2 (SEQ ID NO:3), or a hybridizable fragment thereof, undermoderately stringent conditions; more preferably, it will hybridizeunder high stringency conditions.

Alternatively, the presence of the gene may be detected by assays basedon the physical, chemical, or immunological properties of its expressedproduct. For example, cDNA clones, or DNA clones which hybrid-select theproper mRNAs, can be selected which produce a protein that, e.g., hassimilar or identical electrophoretic migration, isoelectric focusingbehavior, proteolytic digestion maps, tyrosine phosphatase activity orantigenic properties as known for the present modulator peptides. Forexample, the antibodies of the instant invention can conveniently beused to screen for homologs of modulator peptides from other sources.

A gene encoding a modulator peptide of the invention can also beidentified by mRNA selection, i.e., by nucleic acid hybridizationfollowed by in vitro translation. In this procedure, fragments are usedto isolate complementary mRNAs by hybridization. Such DNA fragments mayrepresent available, purified modulator DNA. Immunoprecipitationanalysis or functional assays (e.g., tyrosine phosphatase activity) ofthe in vitro translation products of the products of the isolated mRNAsidentifies the mRNA and, therefore, the complementary DNA fragments,that contain the desired sequences. In addition, specific mRNAs may beselected by adsorption of polysomes isolated from cells to immobilizedantibodies specifically directed against a modulator peptide.

A radiolabeled modulator peptide cDNA can be synthesized using theselected mRNA (from the adsorbed polysomes) as a template. Theradiolabeled mRNA or cDNA may then be used as a probe to identifyhomologous modulator peptide DNA fragments from among other genomic DNAfragments.

Another feature of this invention is the expression of the DNA sequencesdisclosed herein. As is well known in the art, DNA sequences may beexpressed by operatively linking them to an expression control sequencein an appropriate expression vector and employing that expression vectorto transform an appropriate unicellular host.

Such operative linking of a DNA sequence of this invention to anexpression control sequence, of course, includes, if not already part ofthe DNA sequence, the provision of an initiation codon, ATG, in thecorrect reading frame upstream of the DNA sequence.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and Synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol E1, pCR1, pBR322, pMB9, pUC or pUC plasmid derivatives, e.g., pGEXvectors, pmal-c, pFLAG, etc., and their derivatives, plasmids such asRP4; phage DNAs, e.g., the numerous derivatives of phage λ, e.g., NM989,and other phage DNA, e.g., M13 and Filamentous single stranded phageDNA; yeast plasmids such as the 2μ plasmid or derivatives thereof;vectors useful in eukaryotic cells, such as vectors useful in insect ormammalian cells; vectors derived from combinations of plasmids and phageDNAs, such as plasmids that have been modified to employ phage DNA orother expression control sequences; and the like.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a DNA sequence operatively linked to it—may beused in these vectors to express the DNA sequences of this invention.Such useful expression control sequences include, for example, the earlyor late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lacsystem, the trp system, the TAC system, the TRC system, the LTR system,the major operator and promoter regions of phage λ, the control regionsof fd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), thepromoters of the yeast α-mating factors, and other sequences known tocontrol the expression of genes of prokaryotic or eukaryotic cells ortheir viruses, and various combinations thereof.

A wide variety of unicellular host cells are also useful in expressingthe DNA sequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animalcells, such as CHO, R1.1, B-W and L-M cells, African Green Monkey kidneycells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g.,Sf9), and human cells and plant cells in tissue culture.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences ofthis invention. Neither will all hosts function equally well with thesame expression system. However, one skilled in the art will be able toselect the proper vectors, expression control sequences, and hostswithout undue experimentation to accomplish the desired expressionwithout departing from the scope of this invention. For example, inselecting a vector, the host must be considered because the vector mustfunction in it. The vector's copy number, the ability to control thatcopy number, and the expression of any other proteins encoded by thevector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly asregards potential secondary structures. Suitable unicellular hosts willbe selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the DNA sequences to beexpressed, and the ease of purification of the expression products.

Considering these and other factors a person skilled in the art will beable to construct a variety of vector/expression control sequence/hostcombinations that will express the DNA sequences of this invention onfermentation or in large scale animal culture.

In a specific embodiment, an ob fusion protein can be expressed. An obfusion protein comprises at least a functionally active portion of anon-ob protein joined via a peptide bond to at least a functionallyactive portion of an ob polypeptide. The non-ob sequences can be amino-or carboxy-terminal to the ob sequences. More preferably, for stableexpression of a proteolytically inactive ob fusion protein, the portionof the non-ob fusion protein is joined via a peptide bond to the aminoterminus of the ob protein. A recombinant DNA molecule encoding such afusion protein comprises a sequence encoding at least a functionallyactive portion of a non-ob protein joined in-frame to the ob codingsequence, and preferably encodes a cleavage site for a specificprotease, e.g., thrombin or Factor Xa, preferably at the ob-non-objuncture. In a specific embodiment, the fusion protein is expressed inEscherichia coli.

In a specific embodiment, infra, vectors were prepared to express themurine and human ob genes, with and without the codon for gln-49, inbacterial expression systems as fusion proteins. The ob gene is preparedwith an endonuclease cleavage site, e.g., using PCR and novel primers.It is desirable to confirm sequences generated by PCR, since theprobability of including a point mutation is greater with thistechnique. A plasmid containing a histidine tag (HIS-TAG) and aproteolytic cleavage site is used. The presence of the histidine makespossible the selective isolation of recombinant proteins on aNi-chelation column, or by affinity purification. The proteolyticcleavage site, in a specific embodiment, infra, a thrombin cleavagesite, is engineered so that treatment with the protease, e.g., thrombin,will release the full length mature (i.e., lacking a signal sequence) obpolypeptide.

In another aspect, the gex vector (Smith and Johnson, 1988, Gene67:31-40) can be used. This vector fuses the schistosoma japonicumglutathionine S-transferase cDNA to the sequence of interest. Bacterialproteins are harvested and recombinant proteins can be quickly purifiedon a reduced glutathione affinity column. The GST carrier cansubsequently be cleaved from fusion proteins by digestion withsite-specific proteases. After cleavage, the carrier and uncleavedfusion protein can be removed by absorption on glutathione agarose.Difficulty with the system occasionally arises when the encoded proteinis insoluble in aqueous solutions.

In addition to the specific example, the present inventors contemplateuse of baculovirus, mammalian, and yeast expression systems to expressthe ob protein. For example, in baculovirus expression systems, bothnon-fusion transfer vectors, such as but not limited to pVL941 (BamHIcloning site; Summers), pVL1393 (BamHI, SmaI, XbaI, EcoRI, NotI, XmalII,BglII, and PstI cloning site; Invitrogen), pVL1392 (BglII, PstI, NotI,XmalII, EcoRI, XbaI, SmaI, and BamHI cloning site; Summers andInvitrogen), and pBlueBaclII (BamHI, BglII, PstI, NcoI, and HindIIIcloning site, with blue/white recombinant screening possible;Invitrogen), and fusion transfer vectors, such as but not limited topAc700 (BamHI and KpnI cloning site, in which the BamHI recognition sitebegins with the initiation codon; Summers), pAc701 and pAc702 (same aspAc700, with different reading frames), pAc360 (BamHI cloning site 36base pairs downstream of a polyhedrin initiation codon;Invitrogen(195)), and pBlueBacHisA, B, C (three different readingframes, with BamHI, BglII, PstI, NcoI, and HindIII cloning site, anN-terminal peptide for ProBond purification, and blue/white recombinantscreening of plaques; Invitrogen (220)).

Mammalian expression vectors contemplated for use in the inventioninclude vectors with inducible promoters, such as dihydrofolatereductase (DHFR), e.g., any expression vector with a DHFR expressionvector, or a DHFR/methotrexate co-amplification vector, such as pED(PstI, SalI, SbaI, SmaI, and EcoRI cloning site, with the vectorexpressing both the cloned gene and DHFR; see Kaufman, Current Protocolsin Molecular Biology, 16.12, 1991). Alternatively, a glutaminesynthetase/methionine sulfoximine co-amplification vector, such as pEE14(HindIII, XbaI, SmaI, SmaI, EcoRI, and BclI cloning site, in which thevector expresses glutamine synthase and the cloned gene; Celltech). Inanother embodiment, a vector that directs episomal expression undercontrol of Epstein Barr Virus (EBV) can be used, such as pREP4 (BamHII,SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnI cloning site,constitutive RSV LTR promoter, hygromycin selectable marker;Invitrogen), pCEP4 (BamHI, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII,and KpnI cloning site, constitutive hCMV immediate early gene,hygromycin selectable marker; Invitrogen), pMEP4 (KpnI, PvuI, NheI,HindIII, NotI, XhoI, SfiI, BamHI cloning site, induciblemethallothionein IIa gene promoter, hygromycin selectable marker:Invitrogen), pREP8 (BamHI, XhoI, NotI, HindIII, NheI, and KpnI cloningsite, RSV LTR promoter, histidinol selectable marker; Invitrogen), pREP9(KpnI, NheI, HindII, NotI, XhoI, SfiI, and BamHI cloning site, RSV LTRpromoter, G418 selectable marker; Invitrogen), and pEBVHis (RSV LTRpromoter, hygromycin selectable marker, N-terminal peptide purifiablevia ProBond resin and cleaved by enterokinase; Invitrogen). Selectablemammalian expression vectors for use in the invention include pRc/CMV(HindIII, BstXI, NotI, SbaI, and ApaI cloning site, G418 selection;Invitrogen), pRc/RSV (HindIII, SpeI, BstXI, NotI, XbaI cloning site,G418 selection; Invitrogen), and others. Vaccinia virus mammalianexpression vectors (see, Kaufman, supra) for use according to theinvention include but are not limited to pSC11 (SmaI cloning site, TK-and β-gal selection), pMJ601 (SalII, SmaI, AflI, NarI, BspMII, BamHI,ApaI, NheI, SacII, KpnI, and HindIII cloning site; TK- and β-galselection), and pTKgptFIS (EcoRI, PstI, SalI, AccI, HindII, SbaI, BamHI,and HpA cloning site, TK or XPRT selection).

Yeast expression systems can also be used according to the invention toexpress ob polypeptide. For example, the non-fusion pYES2 vector (XbaI,SphI, ShoI, NotI, GstXI, EcoRI, BstXI, BamHI, SacI, KpnI, and HindIIIcloning sit; Invitrogen) or the fusion pYESHisA, B, C (XbaI, SphI, ShoI,NotI, BstXI, EcoRI, BamHI, SacI, KpnI, and HindIII cloning site,N-terminal peptide purified with ProBond resin and cleaved withenterokinase; Invitrogen), to mention just two, can be employedaccording to the invention.

It is further intended that body weight modulator peptide analogs may beprepared from nucleotide sequences derived within the scope of thepresent invention. Analogs, such as fragments, may be produced, forexample, by pepsin digestion of weight modulator peptide material. Otheranalogs, such as muteins, can be produced by standard site-directedmutagenesis of weight modulator peptide coding sequences. Analogsexhibiting “weight modulator activity” such as small molecules, whetherfunctioning as promoters or inhibitors, may be identified by known invivo and/or in vitro assays.

As mentioned above, a DNA sequence encoding weight modulator peptides asdisclosed herein can be prepared synthetically rather than cloned. TheDNA sequence can be designed with the appropriate codons for the weightmodulator peptide amino acid sequences. In general, one will selectpreferred codons for the intended host if the sequence will be used forexpression. The complete sequence is assembled from overlappingoligonucleotides prepared by standard methods and assembled into acomplete coding sequence. See, e.g., Edge, Nature, 292:756 (1981);Nambair et al., Science, 223:1299 (1984); Jay et al., J. Biol. Chem.,259:6311 (1984).

Synthetic DNA sequences allow convenient construction of genes whichwill express weight modulator analogs or “muteins”. Alternatively, DNAencoding muteins can be made by site-directed mutagenesis of nativemodulator genes or cDNAs, and muteins can be made directly usingconventional polypeptide synthesis.

A general method for site-specific incorporation of unnatural aminoacids into proteins is described in Christopher J. Noren, Spencer J.Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, Science,244:182-188 (April 1989). This method may be used to create analogs ofthe ob polypeptide with unnatural amino acids.

The present invention extends to the preparation of antisensenucleotides and ribozymes that may be used to interfere with theexpression of the weight modulator proteins at the translational level.This approach utilizes antisense nucleic acid and ribozymes to blocktranslation of a specific mRNA, either by masking that mRNA with anantisense nucleic acid or cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (See Weintraub, 1990;Marcus-Sekura, 1988). In the cell, they hybridize to that mRNA, forminga double stranded molecule. The cell does not translate an mRNA in thisdouble-stranded form. Therefore, antisense nucleic acids interfere withthe expression of mRNA into protein. Oligomers of about fifteennucleotides and molecules that hybridize to the AUG initiation codonwill be particularly efficient, since they are easy to synthesize andare likely to pose fewer problems than larger molecules when introducingthem into weight modulator peptide-producing cells. Antisense methodshave been used to inhibit the expression of many genes in vitro(Marcus-Sekura, 1988; Hambor et al., 1988).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single stranded RNA molecules in a manner somewhatanalogous to DNA restriction endonucleases. Ribozymes were discoveredfrom the observation that certain mRNAs have the ability to excise theirown introns. By modifying the nucleotide sequence of these RNAs,researchers have been able to engineer molecules that recognize specificnucleotide sequences in an RNA molecule and cleave it (Cech, 1988.).Because they are sequence-specific, only mRNAs with particular sequencesare inactivated.

Investigators have identified two types of ribozymes, Tetrahymena-typeand “hammerhead”-type (Hasselhoff and Gerlach, 1988). Tetrahymena-typeribozymes recognize four-base sequences, while “hammerhead”-typerecognize eleven- to eighteen-base sequences. The longer the recognitionsequence, the more likely it is to occur exclusively in the target mRNAspecies. Therefore, hammerhead-type ribozymes are preferable toTetrahymena-type ribozymes for inactivating a specific mRNA species, andeighteen base recognition sequences are preferable to shorterrecognition sequences.

The DNA sequences described herein may thus be used to prepare antisensemolecules against, and ribozymes that cleave mRNAs for weight modulatorproteins and their ligands.

The present invention also relates to a variety of diagnosticapplications, including methods for detecting the presence of conditionsand/or stimuli that impact abnormalities in body weight or adiposity, byreference to their ability to elicit the activities which are mediatedby the present weight modulators. As mentioned earlier, the weightmodulator peptides can be used to produce antibodies to themselves by avariety of known techniques, and such antibodies could then be isolatedand utilized as in tests for the presence of particular transcriptionalactivity in suspect target cells.

Antibody(ies) to the body weight modulators, i.e., the ob polypeptide,can be produced and isolated by standard methods including the wellknown hybridoma techniques. For convenience, the antibody(ies) to theweight modulators will be referred to herein as Ab₁ and antibody(ies)raised in another species as Ab₂.

According to the invention, ob polypeptide produced recombinantly or bychemical synthesis, and fragments or other derivatives or analogsthereof, including fusion proteins, may be used as an immunogen togenerate antibodies that recognize the ob polypeptide. Such antibodiesinclude but are not limited to polyclonal, monoclonal, chimeric, singlechain, Fab fragments, and an Fab expression library.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to ob polypeptide a recombinant PTP or derivativeor analog thereof. For the production of antibody, various host animalscan be immunized by injection with the ob polypeptide, or a derivative(e.g., fragment or fusion protein) thereof, including but not limited torabbits, mice, rats, sheep, goats, etc. In one embodiment, the obpolypeptide or fragment thereof can be conjugated to an immunogeniccarrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin(KLH). Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward the obpolypeptide, or fragment, analog, or derivative thereof, any techniquethat provides for the production of antibody molecules by continuouscell lines in culture may be used. These include but are not limited tothe hybridoma technique originally developed by Kohler and Milstein(1975, Nature 256:495-497), as well as the trioma technique, the humanB-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72),and the EBV-hybridoma technique to produce human monoclonal antibodies(Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). In an additional embodiment of the invention,monoclonal antibodies can be produced in germ-free animals utilizingrecent technology (PCT/US90/02545). According to the invention, humanantibodies may be used and can be obtained by using human hybridomas(Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or bytransforming human B cells with EBV virus in vitro (Cole et al., 1985,in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).In fact, according to the invention, techniques developed for theproduction of “chimeric antibodies” (Morrison et al., 1984, J.Bacteriol. 159-870; Neuberger et al., 1984, Nature 312:604-608; Takedaet al., 1985, Nature 314:452-454) by splicing the genes from a mouseantibody molecule specific for an ob polypeptide together with genesfrom a human antibody molecule of appropriate biological activity can beused; such antibodies are within the scope of this invention. Such humanor humanized chimeric antibodies are preferred for use in therapy ofhuman diseases or disorders (described infra), since the human orhumanized antibodies are much less likely than xenogenic antibodies toinduce an immune response, in particular an allergic response,themselves.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce ob polypeptide-specific single chain antibodies. An additionalembodiment of the invention utilizes the techniques described for theconstruction of Fab expression libraries (Huse et al., 1989, Science246:1275-1281) to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity for an ob polypeptide, or itsderivatives, or analogs.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbent assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention. For example, to select antibodies which recognize aspecific epitope of an ob polypeptide, one may assay generatedhybridomas for a product which binds to an ob polypeptide fragmentcontaining such epitope. For selection of an antibody specific to an obpolypeptide from a particular species of animal, one can select on thebasis of positive binding with ob polypeptide expressed by or isolatedfrom cells of that species of animal.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the ob polypeptide, e.g.,for Western blotting, imaging ob polypeptide in situ, measuring levelsthereof in appropriate physiological samples, etc.

In a specific embodiment, antibodies that agonize or antagonize theactivity of ob polypeptide can be generated. Such antibodies can betested using the assays described infra for identifying ligands.

Immortal, antibody-producing cell lines can also be created bytechniques other than fusion, such as direct transformation of Blymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980); Hammerlinget al., “Monoclonal Antibodies And T-cell Hybridomas” (1981); Kennett etal., “Monoclonal Antibodies” (1980); see also U.S. Pat. Nos. 4,341,761;4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500;4,491,632; 4,493,890.

In a specific embodiment, antibodies are developed by immunizing rabbitswith synthetic peptides predicted by the protein sequence or withrecombinant proteins made using bacterial expression vectors. The choiceof synthetic peptides is made after careful analysis of the predictedprotein structure, as described above. In particular, peptide sequencesbetween putative cleavage sites are chosen. Synthetic peptides areconjugated to a carrier such as KLH hemocyanin or BSA using carbodiimideand used in Freunds adjuvant to immunize rabbits. In order to preparerecombinant protein, the gex vector can be used to express thepolypeptide (Smith and Johnson, supra). Alternatively, one can use onlyhydrophilic domains to generate the fusion protein. The expressedprotein will be prepared in quantity and used to immunize rabbits inFreunds adjuvant.

The presence of weight modulator in cells can be ascertained by theusual immunological procedures applicable to such determinations. Anumber of useful procedures are known. Three such procedures which areespecially useful utilize either the receptor recognition factor labeledwith a detectable label, antibody Ab₁ labeled with a detectable label,or antibody Ab₂ labeled with a detectable label. The procedures may besummarized by the following equations wherein the asterisk indicatesthat the particle is labeled, and “WM” stands for the weight modulator:WM*+Ab ₁=WM*Ab ₁  A.WM+Ab*=WMAb ₁*  B.WM+Ab ₁ +Ab ₂*=WMAb ₁ Ab ₂*  C.

The procedures and their application are all familiar to those skilledin the art and accordingly may be utilized within the scope of thepresent invention. The “competitive” procedure, Procedure A, isdescribed in U.S. Pat. Nos. 3,654,090 and 3,850,752. Procedure C, the“sandwich” procedure, is described in U.S. Pat. Nos. RE 31,006 and4,016,043. Still other procedures are known such as the “doubleantibody”, or “DASP” procedure.

In each instance, the weight modulators form complexes with one or moreantibody(ies) or binding partners and one member of the complex islabeled with a detectable label. The fact that a complex has formed and,if desired, the amount thereof, can be determined by known methodsapplicable to the detection of labels.

It will be seen from the above, that a characteristic property of Ab₂ isthat it will react with Ab₁. This is because Ab₁ raised in one mammalianspecies has been used in another species as an antigen to raise theantibody Ab₂. For example, Ab₂ may be raised in goats using rabbitantibodies as antigens. Ab₂ therefore would be anti-rabbit antibodyraised in goats. For purposes of this description and claims, Ab₁ willbe referred to as a primary or anti-weight modulator antibody, and Ab₂will be referred to as a secondary or anti-Ab₁ antibody.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others.

A number of fluorescent materials are known and can be utilized aslabels. These include, for example, fluorescein, rhodamine and auramine.A particular detecting material is anti-rabbit antibody prepared ingoats and conjugated with fluorescein through an isothiocyanate.

The weight modulators or their binding partners can also be labeled witha radioactive element or with an enzyme. The radioactive label can bedetected by any of the currently available counting procedures. Thepreferred isotope may be selected from ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr,⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re.

Enzyme labels are likewise useful, and can be detected by any of thepresently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques. Theenzyme is conjugated to the selected particle by reaction with bridgingmolecules such as carbodiimides, diisocyanates, glutaraldehyde and thelike. Many enzymes which can be used in these procedures are known andcan be utilized. The preferred are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090;3,850,752; and 4,016,043 are referred to by way of example for theirdisclosure of alternate labeling material and methods.

A particular assay system that is to be utilized in accordance with thepresent invention, is known as a receptor assay. In a receptor assay,the material to be assayed is appropriately labeled and then certaincellular test colonies are inoculated with a quantity of both thelabeled and unlabeled material after which binding studies are conductedto determine the extent to which the labeled material binds to the cellreceptors. In this way, differences in affinity between materials can beascertained.

Accordingly, a purified quantity of the weight modulator may beradiolabeled and combined, for example, with antibodies or otherinhibitors thereto, after which binding studies would be carried out.Solutions would then be prepared that contain various quantities oflabeled and unlabeled uncombined weight modulator, and cell sampleswould then be inoculated and thereafter incubated. The resulting cellmonolayers are then washed, solubilized and then counted in a gammacounter for a length of time sufficient to yield a standard error of<5%. These data are then subjected to Scatchard analysis after whichobservations and conclusions regarding material activity can be drawn.While the foregoing is exemplary, it illustrates the manner in which areceptor assay may be performed and utilized, in the instance where thecellular binding ability of the assayed material may serve as adistinguishing characteristic. In turn, a receptor assay will beparticularly useful in the identification of the specific receptors tothe present modulators, such as the receptor present on db.

A further assay useful and contemplated in accordance with the presentinvention is known as a “cis/trans” assay. Briefly, this assay employstwo genetic constructs, one of which is typically a plasmid thatcontinually expresses a particular receptor of interest when transfectedinto an appropriate cell line, and the second of which is a plasmid thatexpresses a reporter such as luciferase, under the control of areceptor/ligand complex. Thus, for example, if it is desired to evaluatea compound as a ligand for a particular receptor, one of the plasmidswould be a construct that results in expression of the receptor in thechosen cell line, while the second plasmid would possess a promoterlinked to the luciferase gene in which the response element to theparticular receptor is inserted. If the compound under test is anagonist for the receptor, the ligand will complex with the receptor, andthe resulting complex will bind the response element and initiatetranscription of the luciferase gene. The resulting chemiluminescence isthen measured photometrically, and dose response curves are obtained andcompared to those of known ligands. The foregoing protocol is describedin detail in U.S. Pat. No. 4,981,784 and PCT International PublicationNo. WO 88/03168, for which purpose the artisan is referred.

In a further embodiment of this invention, commercial test kits suitablefor use by a medical specialist may be prepared to determine thepresence or absence of predetermined transcriptional activity orpredetermined transcriptional activity capability in suspected targetcells. In accordance with the testing techniques discussed above, oneclass of such kits will contain at least the labeled weight modulator orits binding partner, for instance an antibody specific thereto, anddirections, of course, depending upon the method selected, e.g.,“competitive”, “sandwich”, “DASP” and the like. The kits may alsocontain peripheral reagents such as buffers, stabilizers, etc.

Accordingly, a test kit may be prepared for the demonstration of thepresence or capability of cells for predetermined transcriptionalactivity, comprising:

-   -   (a) a predetermined amount of at least one labeled        immunochemically reactive component obtained by the direct or        indirect attachment of the present weight modulator or a        specific binding partner thereto, to a detectable label;    -   (b) other reagents; and    -   (c) directions for use of said kit.

More specifically, the diagnostic test kit may comprise:

-   -   (a) a known amount of the weight modulator as described above        (or a binding partner) generally bound to a solid phase to form        an immunosorbent, or in the alternative, bound to a suitable        tag, or plural such end products, etc. (or their binding        partners) one of each;    -   (b) if necessary, other reagents; and    -   (c) directions for use of said test kit.

In a further variation, the test kit may be prepared and used for thepurposes stated above, which operates according to a predeterminedprotocol (e.g. “competitive”, “sandwich”, “double antibody”, etc.), andcomprises:

-   -   (a) a labeled component which has been obtained by coupling the        weight modulator to a detectable label;    -   (b) one or more additional immunochemical reagents of which at        least one reagent is a ligand or an immobilized ligand, which        ligand is selected from the group consisting of:        -   (i) a ligand capable of binding with the labeled component            (a);        -   (ii) a ligand capable of binding with a binding partner of            the labeled component (a);        -   (iii) a ligand capable of binding with at least one of the            component(s) to be determined; and        -   (iv) a ligand capable of binding with at least one of the            binding partners of at least one of the component(s) to be            determined; and    -   (c) directions for the performance of a protocol for the        detection and/or determination of one or more components of an        immunochemical reaction between the weight modulator and a        specific binding partner thereto.

In accordance with the above, an assay system for screening potentialdrugs effective to mimic or antagonize the activity of the weightmodulator may be prepared. The weight modulator may be introduced into atest system, and the prospective drug may also be introduced into theresulting cell culture, and the culture thereafter examined to observeany changes in the activity of the cells, due either to the addition ofthe prospective drug alone, or due to the effect of added quantities ofthe known weight modulator.

As stated earlier, the molecular cloning of the ob gene described hereinhas led to the identification of a class of materials that function onthe molecular level to modulate mammalian body weight. The discovery ofthe modulators of the invention has important implications for thediagnosis and treatment of nutritional disorders including, but notlimited to, obesity, weight loss associated with cancer and thetreatment of diseases associated with obesity such as hypertension,heart disease and Type II diabetes. In addition, there are potentialagricultural uses for the gene product in cases where one might wish tomodulate the body weight of domestic animals. Finally, to the extentthat one or more of the modulators of the invention are secretedmolecules, they can be used biochemically to isolate their receptorusing the technology of expression cloning. The discussion that followswith specific reference to the ob gene bears general applicability tothe class of modulators that a part of the present invention, and istherefore to be accorded such latitude and scope of interpretation.

Therapeutic Implications

In the simplest analysis the ob gene determines body weight in mammals,in particular mice and man. The ob gene and, correspondingly, cognatemolecules, appear to be part of a signaling pathway by which adiposetissue communicates with the brain and the other organs. It is believedthat the ob polypeptide is itself a signaling molecule, i.e., a hormone.Alternatively ob may be responsible for the generation of a metabolicsignal, e.g., an enzyme that catalyzes the synthesis of a peptide orsteroid hormone. The most important piece of information fordistinguishing between these possibilities or considering alternativehypothesis, is the complete DNA sequence of the RNA and its predictedprotein sequence. Irrespective of its biochemical function the geneticdata suggest that increased activity of ob would result in weight losswhile decreased activity would be associated with weight gain. The meansby which the activity of ob can be modified so as to lead to atherapeutic effect depends on its biochemical function.

Administration of recombinant ob polypeptide is believed to result inweight loss. Recombinant protein can be prepared using standardbacterial and/or mammalian expression vectors, all as stated in detailearlier herein. Reduction of ob polypeptide activity (by developingantagonists, inhibitors, or antisense molecules) should result in weightgain as might be desirable for the treatment of the weight lossassociated with cancer, AIDS or anorexia nervosa. Modulation of obactivity can be useful for reducing body weight (by increasing itsactivity) or increasing body weight (by decreasing its activity).

For example, the ob gene could be introduced into human fat cells todevelop gene therapy for obesity. Such therapy would be expected todecrease body weight. Conversely, introduction of antisense constructsinto human fat cells would reduce the levels of active ob polypeptideand would be predicted to increase body adiposity.

If ob is an enzyme, strategies have begun to be developed for theidentification of the substrate and product of the catalyzed reactionthat would make use of the recombinant protein. The rationale for thisstrategy is as follows: If ob is an enzyme that catalyzes a particularreaction in adipose tissue, then fat cells from ob mice should have highlevels of the substrate and very little product. Since it ishypothesized that db mice are resistant to the product of this reaction,fat cells from db mice should have high levels of the reaction product.Thus, comparisons of lipid and peptide extracts of ob and db adiposetissue using gas chromatography or other chromatographic methods shouldallow the identification of the product and substrate of the keychemical reaction. The prediction would be that the recombinant obprotein would catalyze this reaction. The product of this reaction wouldthen be a candidate for a signaling molecule that modulates body weight.

The functional activity of the ob polypeptide, and therapeutic usesthereof, can be determined using transgenic mice. Candidate genes lessthan ˜40 kb can be used in complementation studies employing transgenicmice. Transgenic vectors, including viral vectors, or cosmid clones (orphage clones) corresponding to the wild type locus of candidate gene,can be constructed using the isolated YACs as starting material. Cosmidsmay be introduced into transgenic mice using published procedures(Jaenisch, Science 240, 1468-1474, 1988). The constructs are introducedinto fertilized eggs derived from an intercross between F1 progeny of aC57BL/6J ob/ob X DBA intercross. These crosses require the use ofC57BL/6J ob/ob ovarian transplants to generate the F1 animals. DBA/2Jmice are used as the counterstrain because they have a nonagouti coatcolor which is important when using the ovarian transplants. Genotype atthe ob loci in cosmid transgenic animals can be determined by typinganimals with tightly linked RFLPs or microsatellites which flank themutation and which are polymorphic between the progenitor strains.Complemention will be demonstrated when a particular construct renders agenetically obese F2 animal (as scored by RFLP analysis) lean andnondiabetic. Under these circumstances, final proof of complementationwill require that the ob/ob or db/db animal carrying the transgene bemated to the ob/ob or db/db ovarian transplants. In this cross, all N2animals which do not carry the transgene will be obese and insulinresistant/diabetic, while those that do carry the transgene will be leanand have normal glucose and insulin concentrations in plasma. In agenetic sense, the transgene acts as a suppressor mutation.Alternatively, ob genes can be tested by examining their phenotypiceffects when express in antisense orientation in wild-type animals. Inthis approach, expression of the wild type allele is suppressed, whichleads to a mutant phenotype. RNARNA duplex formation (antisensesense)prevents normal handling of mRNA, resulting in partial or completeelimination of wild-type gene effect. This technique has been used toinhibit Tk synthesis in tissue culture and to produce phenotypes of theKruppel mutation in Drosophila, and the shiverer mutation in mice (Izantand Weintraub, Cell 36, 1007-1015, 1984; Green et al., Annu. Rev.Biochem. 55,569-597, 1986; Katsuki et al., Science 241, 593-595, 1988).An important advantage of this approach is that only a small portion ofthe gene need be expressed for effective inhibition of expression of theentire cognate mRNA. The antisense transgene will be placed undercontrol of its own promoter or another promoter expressed in the correctcell type, and placed upstream of the SV40 poly A site. This transgenewill be used to make transgenic mice. Transgenic mice will also be matedovarian transplants to test whether ob heterozygotes are more sensitiveto the effects of the antisense construct.

In the long term, the elucidation of the biochemical function of the obprotein/gene product should also be useful for identifying smallmolecule agonists and antagonists that affect its activity.

Diagnostic Implications

The human cDNA clones that have recently been isolated have beensequenced as presented herein. This facilitates the determination of thecomplete sequence of the human gene. It is also proposed to generate DNAsequences from the introns of the human ob gene. This will make itpossible to generate DNA sequences from the introns of the human obgene, and thereafter to PCR amplify the coding sequence of the ob genefrom human genomic DNA so as to identify mutations or allelic variantsof the ob gene, all in accordance with protocols described in detailearlier herein.

The current hypothesis is that heterozygous mutations in the ob genewill be associated with mild/moderate obesity while homozygous mutationswould be associated with several DNA sequence based diagnostic testsobesity. If this is true, it would allow the ascertainment of people atrisk for the development of obesity and make possible the application ofdrug treatment and/or lifestyle changes before an increased body weightis full developed.

The ob gene may also be useful diagnostically for measurements of itsencoded RNA and protein in nutritional disorders. It will be ofimportance to know, in a particular nutritional disorder, whether ob RNAand/or protein is unregulated or downregulated. Thus, if an obese personhas increased levels of ob we would conclude that the problem isdownstream of ob, while if ob is reduced we would conclude thatinappropriately low levels of ob may be cause of obesity (whether or notthe defect is in the ob gene). Conversely, if a cancer or HIV patientwho lost weight had elevated levels of ob, we might conclude thatinappropriately high expression of ob is responsible for the weightloss.

The cloned human cDNA will be of use for the measurement of the levelsof human ob RNA. In addition, recombinant human protein will be preparedand used to develop radioimmunoassays to enable us to measure the fatand perhaps plasma levels of the ob protein.

Agricultural Applications

The ob gene can also be isolated from domestic animals, and thecorresponding ob polypeptide obtained thereby. In a specific example,infra, the a probe derived from the murine ob gene hybridizes tocorresponding homologous coding sequences from a large number of speciesof animals. As discussed for human therapies, recombinant proteins canalso be prepared and administered to domestic animals. Administration ofthe polypeptide is desired to produce leaner food animals, such as beefcattle, swine, poultry, sheep, etc. Preferably, an autologous obpolypeptide is administered, although the invention contemplatesadministration of anti-autologous polypeptide as well. Since the obpolypeptide consists of approximately 160 amino acid residues, it maynot be highly immunogenic. Thus, administration of non-autologouspolypeptide may not result in an immune response.

Alternatively, the introduction of the cloned genes into transgenicdomestic animals would allow one to potentially decrease body weight andadiposity by overexpressing an ob transgene. The simplest means ofachieving this would be to target an ob transgene to fat using its ownor another fat specific promoter. Increases in body fat might bedesirable in other circumstances such as for the development of Kobebeef or fatty liver to make foie gras. This could be accomplished bytargeting an antisense ob transgene to fat, or by using gene knockouttechnology.

Conversely, where an increase in body weight at percentage of fat isdesired, an inhibitor or antagonist of the ob polypeptide can beadministered. Such inhibitors or antagonists include, but are notlimited to, antibodies reactive with the polypeptide, and fragments ofthe polypeptide that bind but do not activate the ob receptor.

The ob Receptor

Development of small molecule agonists and antagonists of the ob factorwill be greatly facilitated by the isolation of its receptor. This canbe accomplished by preparing active ob polypeptide and using it toscreen an expression library using standard methodology. Receptorbinding in the expression library can be tested by administeringrecombinant polypeptide prepared using either bacterial or mammalianexpression vectors, and observing the effects of short term andcontinuous administration of the recombinant polypeptide on the cells ofthe expression library, or by directly detecting binding of obpolypeptide to the cells.

As it is presently believed that the ob receptor is likely to be locatedin the hypothalamus and perhaps liver, preferably cDNA libraries fromthese tissues will be constructed in standard expression cloningvectors. These cDNA clones would next be introduced into COS cells aspools and the resulting transformants would be screened with activeligand to identify COS cells expressing the ob receptor. Positive clonescan then be isolated so as to recover the cloned receptor. The clonedreceptor would be used in conjunction with the ob ligand (assuming it isa hormone) to develop the necessary components for screening of smallmolecule modulators of ob.

Once a recombinant which expresses the ob receptor gene sequence isidentified, the recombinant ob receptor can be analyzed. This isachieved by assays based on the physical or functional properties of theob receptor, including radioactive labelling of the receptor followed byanalysis by gel electrophoresis, immunoassay, ligand binding, etc.Furthermore, antibodies to the ob receptor could be generated asdescribed above.

The structure of the ob receptor can be analyzed by various methodsknown in the art. Preferably, the structure of the various domains,particularly the ob binding site, is analyzed. Structural analysis canbe performed by identifying sequence similarity with other knownproteins, particular hormone and protein receptors. The degree ofsimilarity (or homology) can provide a basis for predicting structureand function of the ob receptor, or a domain thereof. In a specificembodiment, sequence comparisons can be performed with sequences foundin GenBank, using, for example, the FASTA and FASTP programs (Pearsonand Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-48).

The protein sequence can be further characterized by a hydrophilicityanalysis (e.g., Hopp and Woods, 1981, Proc. Natl. Acad. Sci. U.S.A.78:3824). A hydrophilicity profile can be used to identify thehydrophobic and hydrophilic regions of the ob receptor protein, whichmay in turn indicate extracytoplasmic, membrane binding, andintracytoplasmic regions.

Secondary structural analysis (e.g., Chou and Fasman, 1974, Biochemistry13:222) can also be done, to identify regions of the ob receptor thatassume specific secondary structures.

Manipulation, translation, and secondary structure prediction, as wellas open reading frame prediction and plotting, can also be accomplishedusing computer software programs available in the art.

By providing an abundant source of recombinant ob polypeptide, and theopportunity to isolate the ob receptor, the present invention enablesquantitative structural determination of the active conformation of theob polypeptide and the ob receptor, or domains thereof. In particular,enough material is provided for nuclear magnetic resonance (NMR),infrared (IR), Raman, and ultraviolet (UV), especially circulardichroism (CD), spectroscopic analysis. In particular NMR provides verypowerful structural analysis of molecules in solution, which moreclosely approximates their native environment (Marion et al., 1983,Biochem. Biophys. Res. Comm. 113:967-974; Bar et al., 1985, J. Magn.Reson. 65:355-360; Kimura et al., 1980, Proc. Natl. Acad. Sci. U.S.A.77:1681-1685). Other methods of structural analysis can also beemployed. These include but are not limited to X-ray crystallography(Engstom, A., 1974, Biochem. Exp. Biol. 11:7-13).

More preferably, co-crystals of ob polypeptide and ob receptor can bestudied. Analysis of co-crystals provides detailed information aboutbinding, which in turn allows for rational design of ligand agonists andantagonists. Computer modeling can also be used, especially inconnection with NMR or X-ray methods (Fletterick, R. and Zoller, M.(eds.), 1986, Computer Graphics and Molecular Modeling, in CurrentCommunications in Molecular Biology, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.).

Identification and isolation of a gene encoding an ob receptor of theinvention provides for expression of the receptor in quantities greaterthan can be isolated from natural sources, or in indicator cells thatare specially engineered to indicate the activity of a receptorexpressed after transfection or transformation of the cells. According,in addition to rational design of agonists and antagonists based on thestructure of ob polypeptide, the present invention contemplates analternative method for identifying specific ligands of ob receptor usingvarious screening assays known in the art.

Any screening technique known in the art can be used to screen for obreceptor agonists or antagonists. The present invention contemplatesscreens for small molecule ligands or ligand analogs and mimics, as wellas screens for natural ligands that bind to and agonize or antagonizeactivates ob receptor in vivo.

Knowledge of the primary sequence of the receptor, and the similarity ofthat sequence with proteins of known function, can provide an initialclue as the inhibitors or antagonists of the protein. Identification andscreening of antagonists is further facilitated by determiningstructural features of the protein, e.g., using X-ray crystallography,neutron diffraction, nuclear magnetic resonance spectrometry, and othertechniques for structure determination. These techniques provide for therational design or identification of agonists and antagonists.

Another approach uses recombinant bacteriophage to produce largelibraries. Using the “phage method” (Scott and Smith, 1990, Science249:386-390; Cwirla, et al., 1990, Proc. Natl. Acad. Sci., 87:6378-6382;Devlin et al., 1990, Science, 249:404-406), very large libraries can beconstructed (10⁶-10⁸ chemical entities).

A second approach uses primarily chemical methods, of which the Geysenmethod (Geysen et al., 1986, Molecular Immunology 23:709-715; Geysen etal. 1987, J. Immunologic Method 102:259-274) and the recent method ofFodor et al. (1991, Science 251, 767-773) are examples. Furka et al.(1988, 14th International Congress of Biochemistry, Volume 5, AbstractFR:013; Furka, 1991, Int. J. Peptide Protein Res. 37:487-493), Houghton(U.S. Pat. No. 4,631,211, issued December 1986) and Rutter et al. (U.S.Pat. No. 5,010,175, issued Apr. 23, 1991) describe methods to produce amixture of peptides that can be tested as agonists or antagonists.

In another aspect, synthetic libraries (Needels et al., 1993,“Generation and screening of an oligonucleotide encoded syntheticpeptide library,” Proc. Natl. Acad. Sci. USA 90:10700-4; Lam et al.,International Patent Publication No. WO 92/00252, each of which isincorporated herein by reference in its entirety), and the like can beused to screen for ob receptor ligands according to the presentinvention. With such libraries, receptor antagonists can be detectedusing cell that express the receptor without actually cloning the obreceptor (Lam et al., supra).

Alternatively, assays for binding of soluble ligand to cells thatexpress recombinant forms of the ob receptor ligand binding domain canbe performed. The soluble ligands can be provided readily as recombinantor synthetic ob polypeptide.

The screening can be performed with recombinant cells that express theob receptor, or alternatively, using purified receptor protein, e.g.,produced recombinantly, as described above. For example, the ability oflabeled, soluble or solubilized ob receptor that includes theligand-binding portion of the molecule, to bind ligand can be used toscreen libraries, as described in the foregoing references.

EXAMPLE SECTION

The following outlines the method used to identify the genetic materialthat is exemplary of the present invention. This endeavor comprises foursequential steps; A) Genetic Mapping, B) Physical Mapping, C) CandidateGene Isolation, and D) Mutation detection. Following confirmation thatthe murine gene in object was isolated (Step D), the homologous humangene was sought. The steps are summarized in greater detail, below.

A. Genetic Mapping

The mutation was segregated in genetic crosses and standard linkageanalysis was used to position the mutation relative to RFLPs(restriction fragment length polymorphisms). These data placed the obgene in an ˜5 cM interval on proximal mouse chromosome 6. (5 cM is ameasurement of genetic distance corresponding to 5 apparent geneticcrossovers per 100 animals.) A total of 771 informative meioses weregenerated and used in subsequent genetic mapping (Friedman et al.Genomics 11: 1054-1062, 1991). The genetic loci that were mappedrelative to ob were all previously published. The two closest RFLPsdescribed were defined by probes derived from the carboxypeptidase andmet oncogene genes.

The genetic resolution of the experiments described above was inadequateto clone ob, principally because none of the genetic markers were intight linkage. In order to identify the requisite tightly linked RFLPs,additional probes were isolated and the genetic cross was expanded. Amethod known as chromosome microdissection was used to isolate randompieces of DNA from proximal mouse chromosome 6 (Bahary et al., MammalianGenome 4: 511-515, 1993). Individual cloned probes were tested for tightlinkage to ob. On the basis of these studies one probe, D6Rck13, alsotermed psd3, was selected for further analysis owing to its geneticproximity to ob.

This probe was used to genotype the 771 animals described in Bahary etal. as well as 350 animals derived from an additional cross between obmice and Mus Castaneus mice. On the basis of these data, it wasconcluded that D6Rck13 was ˜0.06 cM distal to ob and was in close enoughproximity to ob to begin cloning efforts. D6Rck13 was recombinant to asingle animal, #167. An additional probe, Pax-4, was identified that was0.12 cM proximal to ob. Pax-4 was recombinant in two animals; #111 and420. Pax-4 is a pseudogene that was previously mapped to proximal mousechromosome 6 by Gruss and co-workers (Gruss et al. Genomics 11:424-434,1991). On this basis, it was determined that the ob gene resides in the˜0.2 cM interval between Pax-4 and D6Rck13. This led to efforts to clonethe interposing DNA in an effort to isolate ob.

B. Physical Mapping

The cloning of the DNA in this interval made use of yeast artificialchromosomes (YACs), a relatively new cloning vector that allows thecloning of long stretches of contiguous DNA often more than 1 millionbase pairs in length.

Firstly, yeast artificial chromosomes were isolated using D6Rck13 andPax-4. This was accomplished by preparing purified DNA probes and usingthem to isolate the corresponding YACs. These YACs (#8, 16, 107 and 24)were isolated and initially characterized, and on the basis of theresulting analyses it was concluded that YAC 16 was the YAC thatextended furthest distally, i.e., closest to oh. The key end of YAC #16was then recovered, and it was determined that this end was closer to obthan Pax-4. This end was termed 16M(+). This conclusion was reachedbecause it was shown that this probe was not recombinant in animal #420(as was Pax-4). This end clone was sequenced and used to develop a PCRassay. This PCR assay was next used to isolate two new YACs, adu andaad, by screening a YAC library. The crucial YAC for subsequent studieswas adu. This YAC was characterized and confirmed to be a non-chimeric370 kB YAC. The distal end of adu, known as adu (picL) was isolated, andit was determined that adu (+) was non recombinant in all the ob progenyof the genetic crosses including animals #111 and 167.

A PCR assay for this segment was developed using eight specific DNAfragments. Using these primers, 100 kb P1 clones were isolated. P1 phageis a cloning vector that can carry 100,000 base pair genomic inserts.The primers were then used in a PCR screen assay to identifycorresponding P1 clones in pools of colonies. Positive pools were thenprobed for specific clones of interest.

As part of the efforts to complete the physical map of ob, the ends ofthe D6Rck13 YAC (YAC #53) were isolated. One of the ends, known as 53Pic1, was used, as well as the key end of YAC aad (known as aad(+)) toisolate additional P1 clones. The ends of these P1 clones werethemselves used to isolate new P1 clones. The DNA sequencing of theseends was performed closing a gap between the 53 and aad YACs, and ˜2.5million base pairs of DNA was cloned that spanned Pax-4, 16M(+), adu(+), aad(Pic1), 53 (Pic1) and D6Rck13. An ˜500 kB subset of this regionwas isolated in P1 clones. By carefully mapping the sites ofrecombination apparent in animals 111 and 167, it was concluded that obwas situated in an ˜400,000 base pair interval that was spanned by acontiguous series of P1 clones. The key P1 clones, 322 and 323, wereamong those selected for further analyses.

The physical map of the portion of the chromosome carrying ob is shownin FIG. 7A. FIG. 7B represents the YAC cloning vectors that contain ob,or regions proximal to the gene.

C. Isolation of Candidate Genes

The method used to isolate genes in this interval was exon trapping(FIG. 7C). This method used a vector (available from Gibco—BRL LifeSciences) to identify exon DNA (i.e., coding sequences) by selecting forfunctional splice acceptor and donor sequences in genomic DNA introducedinto a test construct. Initial attempts at exon trapping were performedusing cosmid subclones derived from YAC #53. These initial efforts wereunsuccessful. Subsequently, these studies were initiated using a subsetof the P1 clones: 322, 323, 324, 325, and 259. The DNA from these P1swere grown and subcloned into the exon trapping vector. The experimentwas repeated using various P1 clones. In these and one subsequent exontrapping experiment, three candidate genes for ob were identified:325-2, 323-8 and a previously cloned gene, Inosine MonophosphateDehydrogenase (IMPDH). The IMPDH gene had been previously cloned but hadnot been mapped, and its proximity to ob was previously unknown. 325-2was subsequently shown to be a testis specific gene, while 323-8 wasshown to encode a rare brain transcript. None of these genes appeared toencode ob.

After three unsuccessful efforts to exon trap the ob gene, anotherattempt was made by preparing DNA from all the P1s from the critical obregion. These included P1s: 258, 259, 322, 323, 324, 325, 498, 499, 500,653, 654 and numerous others.

Thereafter P1s 258, 260, 322, 498 and 499 were subcloned into the exontrapping vector, and subsequently several plates were prepared withbacterial clones, each of which carried a putative exon. Approximately192 clones representing putative ob candidates were obtained. Theseclones were short inserts cloned into the pGem vector.

Each clone was PCR amplified with PCR primers corresponding to plasmidsequences that flanked the insert. The PCR amplification was performeddirectly on the bacteria that carried the plasmid. The reactions wereset up using a Biomek robot. The PCR products were electrophoresed on a1% agarose gel in TBE buffer that contained ethidium bromide (FIG. 8).Based on our previous experience, we found a consistent artifact suchthat many of the isolates contained two trapped exons derived from thevector. We identified the clones both by their size and the fact thathybridization of DNA probes corresponding to this artifact lothybridized to the corresponding bands on a Southern blot of this gel(data not shown). In this way we excluded 185 of the clones from furtherevaluation.

Thus, the 192 exons, a total of seven exons were selected for furtherstudy. The templates for sequencing were prepared and sequencing wasperformed. The results are presented in FIG. 7. The sequences for the 7exons were analyzed and it was found that 4 were identical and one wasan apparent artifact. In particular, clone 1D12 contained the “HIVsequence”, which refers to the so called artifact band. The exontrapping vector includes HIV sequences; a short segment of these vectorsequences corresponds to this artifact. This left three exons forfurther analysis: 1F1, 2G7 and 1H3. 1F1 was eliminated because it mappedoutside the critical region.

PCR primers for 2G7 were selected and synthesized. The primers usedwere:

5′ CCA GGG CAG GAA AAT GTG (SEQ ID NO: 7) (Tm = 60.0) 3′ CAT CCT GGA CTTTCT GGA TAG G (SEQ ID NO: 8) (Tm = 60.0)

These primers amplified genome DNA with PCR conditions as follows: 25-30cycles with 55° annealing×2′, 72° extension×2′, 94° denaturation×1′ instandard PCR buffer. These primers were also used to generate a labeledprobe by including ³²P dCTP in the PCR reaction with a correspondingreduction in the amount of cold dCTP. The sequence of the exon on 2G7was determined, and is shown in FIG. 10 (SEQ ID NO:9). The portions ofthe sequence corresponding to the PCR primers are underlined.

An RT PCR was performed on a variety of tissue RNAs and it was concludedthat 2G7 was expressed exclusively in fat (not shown). Thereafter,³²P-labelled 2G7 was hybridized to a Northern blot of tissue RNAs (FIG.11) and showed that its RNA was expressed at high level in fat tissuebut was either not expressed or expressed at very low levels in allother tissues (where the signals may be the result of fat contaminatingthe tissue preparations). Ten μg of total RNA from each of the tissueslisted was electrophoresed on an agarose gel with formaldehyde. Theprobe was hybridized at 65° in a standard hybridization buffer, RapidHype (Amersham).

The size of the RNA was ˜4.9 kB. At this point 2G7 was considered to bea viable candidate gene for ob and was analyzed further.

D. Mutation Detection

In order to confirm that 2G7 encoded the ob gene, it was necessary todemonstrate differences in the levels of RNA expression of DNA sequenceof this gene in mutant as compared to wild type animals. Two separatemutations of the ob gene are available for study, C57BL/6J ob/ob (1J)and Ckc/Smj ob/ob (2J). These will be referred hereinafter as 1J and 2J,respectively. (Informal nomenclature is used to refer to the mousestrains studied. Throughout this specification and in the drawings, itwill be understood that C57BL/6J refers to C57BL/6J+/+; CKC/smj refersto SM/Ckc-+^(Dac)-+/+; CKC/smj ob/ob refers toSM/Ckc-+^(Dac)-ob^(2J)/ob^(2J).) RNA was prepared from fat tissue thathad been isolated from 1J, 2J, and control animals. Total RNA for eachsample was reverse transcribed using oligo dT and reverse transcriptase.The resulting single stranded cDNA was then PCR amplified either withthe 2G7 primers (conditions shown above) for the lower band orcommercially available actin primers for the upper band. The RT PCRproducts were run on a 1% agarose TBE gel that was stained with ethidiumbromide (FIG. 12). Using RT PCR it was found that 2G7 mRNA was absent in2J mice. 2G7 mRNA was absent, when tested by RT PCR, from fouradditional 2J animals.

This result was confirmed on a Northern blot (FIG. 13). Ten μg of fatcell RNA from each of the strains were run out. The blot was probed withthe 2G7 probe that was PCR labeled, as discussed. Actin is a control forthe amount of RNA loaded. This probe was labeled by PCR amplification ofthe material, i.e., band, in FIG. 11 using ³²P-dCTP in the PCR reaction.The actin signal is fairly similar in all of the samples. The ob signalis absent in brain because the mRNA is specific to fat cells.

The results of the Northern analysis confirm that 2G7 RNA was absent in2J mice. The ob RNA is absent in the CKC/smj ob/ob mice because in thisobese mutant strain the gene is disrupted such that no RNA is made. Inaddition, the level of 2G7 RNA was increased ˜10-20 fold in 1J as wellas db/db fat. These results are compatible with the hypothesis that obeither encodes circulating hormone or is responsible for the generationof a signal from fat cells that modulate body weight. At this point itwas concluded that 2G7 is the ob gene and predicted that 1J mice have apoint mutation, probably a nonsense mutation leading to a prematuretranslation termination.

These Northern results have been replicated using fat cell RNApreparations from four different 2J animals (FIG. 14). In this assay,ap2 is a fat-specific transcript that was used as a control much thesame as actin in FIG. 13. There is no significance to the varyingdensity of the ap2 band. ap2 was labeled by designing PCR primers formthe published ap2 sequence. The RT PCR products of fat cell RNA werethen relabeled using the same protocol for PCR labeling. This analysisdemonstrates the presence of ob mRNA in normal homozygous orheterozygous animals, and its absence from 2J mutant animals.

Using the labeled 2G7 PCR probe, a total of 50 mouse cDNA clones from amurine fat cell λgt11 cDNA library (Clonetech 5′-STRETCH cDNA fromtesticular fat pads of Swiss mice, #ML3005b), and thirty crosshybridizing human cDNA clones from a human fat cell λgt10 cDNA library(Clonetech 5′-STRETCH cDNA from abdomen #HL1108a) were isolated. Libraryscreening was performed using the plague lift procedure. The filtersfrom the plaque lift were denatured using the autoclave method. Thefilters were hybridized in duplicate with the PCR labeled 2G7 probe(Rapid Hybe buffer, 65° C., overnight). After a 2-4 hourprehybridization, the filters were washed in 2×SSC, 2% SDS, twice for 30minutes at 65° C. and exposed to SRy Llim. Duplicate positives wereplaque purified. Plaque purified phage were PCR amplified usingcommercially available vector primers. For example, λgt10 and λgt11. Theresulting PCR products corresponded to the cDNA insert for each phagewith a small amount of vector sequence at either end. The bands were gelpurified and sequenced using the ABI automated sequencer and the vectorprimers to probe the DNA polymerase. Additional sequence information wasgenerated within each clone by synthesizing internal primers derivedfrom the DNA sequence and repeating the DNA sequence reaction.

Sequencing of the coding sequence of these clones is complete (see FIGS.1 and 3, SEQ ID NOS:1 and 2). Sequencing of the adjacent regions iscontinuing, and to date, ˜1600 bp of sequence from five prime end of themurine mRNA has been compiled. The sequence data suggest that the obgene encodes a 160 amino acid protein that has the features of asecreted protein. In addition, the sequence of the homologous human geneis complete (FIGS. 2 and 4, SEQ ID NOS:3 and 4), and extensive homologybetween the mouse and human genes has been demonstrated.

The mutation has been identified in 1J mice. The mutation is G-A basechange that results in an apparent premature stop codon at amino acid108 and in all likelihood accounts for the 1J mutation (FIG. 15) despiteexpression of the ob mRNA (see FIGS. 12 and 13, C57BL/6J ob/ob lanes).

More recently, Southern blots have been used to conclude that the 2Jmutation is the result of a detectable DNA change at the 5′ end of obthat appears to completely abolish RNA expression. The exact nature ofthis possible rearrangement remains to be determined.

A genomic Southern blot of DNA from the CKC/smj (SM/Ckc-+^(Dac)) andC57BL/6J mice using four different restriction endonucleases wasperformed in order to determine whether the mutant ob yielded a uniquefragment pattern (FIG. 16). Approximately 10 μg of DNA (derived fromgenomic DNA prepared from liver, kidney, or spleen) was restrictiondigested with the restriction enzyme indicated. The DNA was thenelectrophoresed in a 1% agarose TBE gel. The DNA was transferred to animobilon membrane and hybridized to the PCR labeled 2G7 probe. The keyband is the uppermost band in the BglII digest for the CKC/smj ob/ob(SM/Ckc-+^(DAC) ob/ob) DNA. This band is of higher molecular weight thanin the other strain, indicating a mutation in this strain.

FIG. 17 is a southern blot of a BglII digest of genomic DNA from theprogeny of an ob^(2J)/+×ob^(2J)/+ cross. Some of the DNAs have only theupper band, some only the lower band, and some have the both bands. Theanimals with only the upper band are allo-obese, i.e., ob^(2J)/ob^(2J).These data show that the polymorphism (i.e., mutation) shown in FIG. 16segregates in a genetic sense.

Genomic DNA was isolated from mouse, rat, rabbit, vole, cat, cow, sheep,pig, human, chicken, eel, and drosophila, and restriction digested withEcoRI. The digests were electrophoresed on 1% agarose TBE gel. DNA wastransferred to an immobilon membrane and probed with the PCR labeled 2G7probe. The filter was hybridized at 65° C. in Rapid Hype Buffer andwashed with 2×SSC, 2% SDS at 65° C. twice for 30 minutes each wash,i.e., there were two buffer changes. These data indicate that ob isconserved among vertebrates (FIG. 18). Note in this regard that there isa 2 (+) signal in eel DNA; eel is a fish.

In summary, available evidence suggests that body weight and adiposityare physiologically controlled. Seven years ago efforts began toidentify two of the key components of this system: the ob and db genes.As shown in this example, the ob gene has now been identified as a fatspecific gene that plays a key role in regulating body weight. Theproduct of this gene, which is most probably a secreted hormone, willhave important implications for the diagnosis and treatment ofnutritional disorders in man and non-human animals.

Example Identification of a Putative Signal Sequence

The putative signal sequence of the full length murine ob gene wasdetermined by application of a computer algorithm to the method of vonHeijne (Nucl. Acids Res. 14, 4683, 1986). Using this technique, the mostprobable signal sequence was identified in the polypeptide coding regioncorresponding to amino acids 9-23, having the sequence:

FLWLWSYLSYVQA ↑ VP (SEQ ID NO: 10)in which the arrow indicates the putative signal sequence cleavage site.

Example Expression of ob in Bacteria

Both murine and human cDNAs encoding ob have been cloned into a pET-15bexpression vector (Novagen). This vector contains a 17 promoter inconjunction with a lac operator, and expresses a fusion proteincontaining a histidine tag (His-Tag) and a thrombin cleavage siteimmediately upstream of the coding sequence insertion site (FIG. 19)(SEQ ID No:11).

The mouse and human cDNAs were modified such that the alanine at the endof the signal sequence was turned into an NdeI site, as was a separatesequence in the 3′ region. Insertion of the NdeI site was accomplishedusing PCR with novel primers:

Mnde 5′ (murine five prime primer): (SEQ ID NO: 12) CTTATGTTCATATGGTGCCG ATCCAGAAAG TC Mnde-3′ (murine three prime primer): (SEQ IDNO: 13) TCCCTCTACA TATGTCTTGG GAGCCTGGTG GC Hnde-5′ (human five primeprimer): (SEQ ID NO: 14) TCTATGTCCA TATGGTGCCG ATCCAAAAAG TCHnde-3′ (human three prime primer): (SEQ ID NO: 15) TTCCTTCCCATATGGTACTC CTTGCAGGAA GA

The primers contain a 6-base pair mismatch in the middle that introducesNdeI restriction sites at each end of the PCR fragment. Phage carryingeither the mouse or human cDNA were PCR amplified using those primers.The PCR product was digested with NdeI and gel purified on a 1% lowmelting point agarose gel. The gel purified bands were subcloned intothe pET vector. The resulting plasmids were sequenced to ensure thatmutations were not introduced during the PCR amplification step ofcloning. To date constructs for the human and mouse cDNA with glutaminehave been prepared; similar constructs are now being made using the sameprimers and methods to introduce the coding sequence without theglutamine (see the next Example).

Example Both Murine and Human ob Genes are Found in Two Isoforms

An unexpected deletion was observed in about one out of three cDNAclones of the human and murine ob gene. In particular, a three base-pairdeletion, corresponding to the glutamine 49 codon, resulted in a deducedamino acid sequence lacking a glutamine residue at position 49 of thefull length murine (FIG. 5; SEQ ID NO:5) and human (FIG. 6; SEQ ID NO:6)polypeptides. This deletion corresponds to nucleotides 260-261-262 fromthe murine cDNA sequence (FIG. 1; SEQ ID NO:1), and to nucleotides182-183-184 on the human sequence (FIG. 2; SEQ ID NO:3).

The missing codon for glutamine 49 in the cDNA sequences immediatelyfollows the 2G7 exon. The sequence of 2G7 corresponds to the sequenceimmediately upstream of the codon for gln-49 in the mouse ob gene(compare FIG. 10 with FIG. 1). We postulate that some of the cDNA lackthe gln-49 CAG codon because this is at a splice acceptor site. Since AGis the actual acceptor site, slippage of the machinery in some caseswould lead to deletion of the CAG codon. This is shown below:

The “ag” in the sequences above corresponds to the assumed intronsequence upstream of the glutamine codon, and AG is the putativealternative splice site.

Example Preparation of Antibodies to the ob Polypeptide

A set of four peptide sequences from the deduced murine ob sequence wereidentified using immunogenicity plot software (GCG Package). The fourcarboxyl terminal peptide fragments are:

(SEQ ID NO: 18): Val-Pro-Ile-Gln-Lys-Val-Gln-Asp-Asp-Thr-Lys-Thr-Leu-Ile-Lys-Thr (SEQ ID NO: 19):Leu-His-Pro-Ile-Leu-Ser-Leu-Ser-Lys-Met-Asp-Gln- Thr-Leu-Ala (SEQ ID NO:20): Ser-Lys-Ser-Cys-Ser-Leu-Pro-Gln-Thr-Ser-Gly-Leu-Gln-Lys-Pro-Glu-ser-Leu-Asp (SEQ ID NO: 21):Ser-Arg-Leu-Gln-Gly-Ser-Leu-Gln-Asp-Ile-Leu-Gln-Gln-Leu-Asp-Val-Ser-Pro-Glu-Cys

These peptides were conjugated to KLH, and the peptide-KLH conjugateswere used to immunize rabbits using standard techniques. Polyclonalantisera specific for each peptide is recovered from the rabbits.

The following is a list of references related to the above disclosureand particularly to the experimental procedures and discussions.

-   Bahary, N.; G. Zorich; J. D. Pachter; R. L. Leibel; and J. M.    Friedman. 1991. Molecular genetic linkage maps of mouse chromosomes    4 and 6. Genomics 11:33-47.-   Bahary, N.; D. McGraw; R. L. Leibel; and J. M. Friedman. 1991.    Chromosomal microdissection of midmouse chromosome 4: Mapping of    microclones relative to the mouse db gene. Submitted.-   Bahary, N.; J. Pachter; R. Felman; R. L. Leibel; K. A. Albright; S.    Cram; and J. M. Friedman. 1991. Molecular mapping of mouse    chromosomes 4 and 6: Use of a flow-sorted Robertsonian chromosome.    Submitted.-   Blank, R.; J. Eppig; F. T. Fiedorek; W. N. Frankel; J. M.    Friedman; K. Huppi; I. Jackson; and B. Mock. 1991. Mouse    chromosome 4. Mammalian Genome 1(suppl): s51-s78.-   Bogardus, C.; Ravussin, E.; Abbot, W.; Zasakzku, J. K.; Young, A.;    Knowler, W. C.; Friedman, J. M.; R. L. Leibel; N. Bahary; D. A.    Siegel; and G. Truett, G. 1991. Genetic analysis of complex    disorders: Molecular mapping of obesity genes in mice and humans    Annals of the New York Academy of Sciences 630:100-115.-   Friedman, J. M.; R. L. Leibel; and N. Bahary. 1991. Molecular    mapping of obesity genes. Mammalian Genome 1:130-144.-   Friedman, J. M.; R. L. Leibel; N. Bahary; and G. Zorich. 1991.    Molecular mapping of the mouse ob mutation. Genomics, (in press).-   Harris, M. I. (1991). Diabetes Care 14 (suppl. 3), 639-648.-   Harris, M. I.; Hadden, W. C.; Knowler, W. C.; and Bennett, P. H.    (1987). Diabetes 36, 523-534.-   Harris, R. B. S. (1990). FASEB J. 4, 3310-3318.-   Jacobowitz, R., and Moll, P. O. (1986). N. Engl. J. Med. 315, 96-100-   Kessey, R. E. (1980). In Obesity, A. Stunkard, eds. (Philadelphia:    W.B. Sauders Co.), pp. 144-166.-   Kessey, R. E., and Pawley, T. L. (1986). Annu. Rev. Psychol. 37,    109-133.22

Leibel, R. L., N. Bahary and J. M. Friedman. 1990. Genetic variation andnutrition in obesity: Approaches to the molecular genetics of obesity.In Genetic variation and Nutrition (Simopoulos, A. P. and Childs, B.,eds.), S. Karger, Basel, pp. 90-101.

-   Siegel, D.; N. G. Irving; J. M. Friedman; and B. J.    Wainwright. 1991. Localization of the cystic fibrosis transmembrane    conductance regulator to mouse chromosome 6. Cytogenetics Cell    Genetics, submitted.-   Truett, G. E.; N. Bahary; J. M. Friedman; and R. L. Leibel. 1991.    The rat obesity fatty (fa) maps to chromosome 5:Evidence for    homology with the mouse gene diabetes (db). Proc. Natl. Acad. Sci.    USA 88:7806-7809.

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allrespects illustrative and not restrictive, the scope of the inventionbeing indicated by the appended Claims, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein.

Various references are cited throughout this specification, each ofwhich is incorporated herein by reference in its entirety.

1. An isolated DNA molecule, which encodes a body weight modulator,selected from the group consisting of: A. the DNA sequence of FIG. 1(SEQ ID NO: 1); B. the DNA sequence of FIG. 2 (SEQ ID NO: 3); and C. aDNA sequence that codes on expression for an amino acid sequence that isselected from the group consisting of SEQ ID NO: 2 and SEQ ID NO:4.